WO2022145623A1 - Glutamate-cysteine ligase variant and method for producing glutathione using same - Google Patents

Glutamate-cysteine ligase variant and method for producing glutathione using same Download PDF

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WO2022145623A1
WO2022145623A1 PCT/KR2021/012176 KR2021012176W WO2022145623A1 WO 2022145623 A1 WO2022145623 A1 WO 2022145623A1 KR 2021012176 W KR2021012176 W KR 2021012176W WO 2022145623 A1 WO2022145623 A1 WO 2022145623A1
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amino acid
protein
variant
microorganism
glutamate
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PCT/KR2021/012176
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French (fr)
Korean (ko)
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김연수
하철웅
양은빈
임영은
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씨제이제일제당 (주)
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Priority to CN202180085919.3A priority Critical patent/CN117460828A/en
Priority to JP2023525461A priority patent/JP2023546971A/en
Priority to AU2021414658A priority patent/AU2021414658A1/en
Priority to EP21915421.8A priority patent/EP4219697A1/en
Priority to CA3197579A priority patent/CA3197579A1/en
Priority to MX2023006959A priority patent/MX2023006959A/en
Publication of WO2022145623A1 publication Critical patent/WO2022145623A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
    • C12Y603/02—Acid—amino-acid ligases (peptide synthases)(6.3.2)
    • C12Y603/02002—Glutamate-cysteine ligase (6.3.2.2)

Definitions

  • the present application relates to a novel glutamate-cysteine ligase variant and a glutathione production method using the same.
  • Glutathione is an organic sulfur compound most commonly present in cells, and is in the form of a tripeptide in which three amino acids of glycine, glutamate, and cysteine are combined.
  • Glutathione exists in the body in two forms: reduced glutathione (GSH) and oxidized glutathione (GSSG).
  • GSH reduced glutathione
  • GSSG oxidized glutathione
  • Reduced glutathione (GSH) which is present in a relatively high proportion under normal circumstances, is mainly distributed in the liver and skin cells of the human body. It plays an important role, such as a whitening action that inhibits the production.
  • glutathione with various functions has been spotlighted as a material in various fields such as pharmaceuticals, health functional foods, and cosmetics, and is also used in the manufacture of flavor materials, food and feed additives. It is known that glutathione has a great effect of increasing the taste of the raw material and maintaining rich taste, and can be used alone or in combination with other substances as a kokumi flavor enhancer. Usually, Kokumi material has a richer feeling than umami material such as nucleic acid and MSG, and is known to be produced by decomposition and aging of protein.
  • the present applicants confirmed that a microorganism introducing a newly developed glutamate-cysteine ligase variant can produce glutathione in high yield, and completed the present application.
  • the present application provides a glutamate-cysteine ligase variant in which the amino acid corresponding to position 653 from the N-terminus of the amino acid sequence of SEQ ID NO: 1 in a protein having glutamate-cysteine ligase activity is substituted with methionine.
  • the present application provides a polynucleotide encoding the variant, and a vector comprising the same.
  • the present application relates to the variant; a polynucleotide encoding the variant; And it provides a microorganism for producing glutathione, including any one or more of the vector containing the polynucleotide.
  • the present application provides a glutathione production method comprising the step of culturing the microorganism.
  • the novel glutamate-cysteine ligase variant of the present application greatly increases glutathione production, it can be usefully used for high glutathione production.
  • the produced glutathione has an antioxidant effect, a detoxification effect, and an immune enhancing effect, and thus can be usefully used in cosmetic compositions, food compositions, feed compositions, pharmaceutical compositions, and preparation thereof.
  • One aspect of the present application includes an amino acid substitution in a protein having glutamate-cysteine ligase activity, wherein the substitution is an amino acid corresponding to the 653th position from the N-terminus of SEQ ID NO: 1 Including that methionine is substituted , glutamate-cysteine ligase (glutamate-cysteine ligase) variants may be provided.
  • the variant may be a protein variant in which glycine, an amino acid corresponding to position 653 from the N-terminus in the glutamate-cysteine ligase amino acid sequence of SEQ ID NO: 1 is substituted with methionine.
  • GCL Glutamate-cysteine ligase
  • Glutamate-cysteine ligase is an enzyme also called “glutamate-cysteine ligase” or “gamma-glutamylcysteine synthetase (GCS)” .
  • Glutamate-cysteine ligase is known to catalyze the following reactions:
  • the amino acid sequence of glutamate-cysteine ligase is an amino acid sequence encoded by the gsh1 gene, and may be referred to as "GSH1 protein" or "glutamate-cysteine ligase".
  • the amino acid sequence constituting the glutamate-cysteine ligase of the present application can be obtained from a known database, GenBank of NCBI.
  • the glutamate-cysteine ligase may be a protein comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 1, but is not limited thereto.
  • the glutamate-cysteine ligase may be derived from Saccharomyces cerevisiae , and in another example, the amino acid corresponding to position 653 in the amino acid sequence of Saccharomyces SEQ ID NO: 1 is It may be glycine.
  • the present invention is not limited thereto, and a sequence having the same glutamate-cysteine ligase activity as the amino acid sequence may be included without limitation.
  • the glutamate-cysteine ligase of the present application has the amino acid sequence of SEQ ID NO: 1 or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology thereto, or It may be a protein comprising an amino acid sequence with identity.
  • a protein having an amino acid sequence in which some sequence is deleted, modified, substituted or added is also included within the scope of the protein subject to mutation of the present application. is self-evident
  • glutamate-cysteine ligase it is defined as a protein including the amino acid sequence of SEQ ID NO: 1, but a meaningless sequence before and after the amino acid sequence of SEQ ID NO: 1 or a naturally occurring mutation, or its potential It is apparent to those skilled in the art that the glutamate-cysteine ligase of the present application corresponds to the case where the mutation (silent mutation) is not excluded, and if it has the same or corresponding activity as the protein consisting of the amino acid sequence of SEQ ID NO: 1.
  • the term “variant” or “modified polypeptide” refers to the recited sequence in which one or more amino acids are conservatively substituted and/or modified. ), but refers to a protein in which the functions or properties of the protein are maintained.
  • the variant is a glutamate-cysteine ligase variant or glutamate-cysteine ligase in which the amino acid corresponding to the 653 position from the N-terminus of SEQ ID NO: 1 among the above-described glutamate-cysteine ligases is substituted with methionine It may be a variant polypeptide having activity.
  • “glutamate-cysteine ligase variant” may also be described as "(mutant) polypeptide having glutamate-cysteine ligase activity" and "GSH1 variant”.
  • the variant differs from the identified sequence by several amino acid substitutions, deletions or additions.
  • Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the protein and evaluating the properties of the modified protein. That is, the ability of the variant may be increased, unchanged, or decreased compared to the native protein.
  • some variants may include variant polypeptides in which one or more portions, such as an N-terminal leader sequence or a transmembrane domain, have been removed.
  • Other variants may include variants in which a portion is removed from the N- and/or C-terminus of the mature protein.
  • variant or “mutant polypeptide” may be used interchangeably with terms such as variant, modified, mutated protein, mutant (in English, modification, modified protein, mutant, mutein, divergent, variant, etc.) , as long as it is a term used in a mutated meaning, it is not limited thereto.
  • the mutant increases the activity of the mutated protein compared to the native wild-type or unmodified protein, or increases the glutathione production compared to the protein before mutation, the natural wild-type polypeptide or the unmodified polypeptide. not limited
  • conservative substitution refers to substituting one amino acid with another amino acid having similar structural and/or chemical properties. Such variants may have, for example, one or more conservative substitutions while still retaining one or more biological activities. Such amino acid substitutions may generally occur based on similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues.
  • amino acids with electrically charged amino acids positively charged (basic) amino acids are arginine, lysine, and histidine
  • negatively charged (acidic) amino acids are glutamic acid and aspartic acid.
  • Amino acids with uncharged side chains are classified as including glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. can do.
  • variants may contain deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide.
  • the polypeptide may be conjugated with a signal (or leader) sequence at the N-terminus of the protein involved in the transfer of the protein either co-translationally or post-translationally.
  • the polypeptide may also be conjugated with other sequences or linkers to enable identification, purification, or synthesis of the polypeptide.
  • the variant may be a glutamate-cysteine ligase (glutamate-cysteine ligase) variant in which the amino acid corresponding to the 653th position from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is substituted with methionine.
  • the variant may be a variant in which glycine corresponding to position 653 in the amino acid sequence of SEQ ID NO: 1 is substituted with methionine, but is not limited thereto.
  • corresponding position refers to an amino acid residue at a listed position in a protein or polypeptide, or an amino acid residue similar to, identical to, or homologous to, a listed residue in a protein or polypeptide.
  • corresponding region generally refers to a similar or corresponding position in a related protein or reference protein.
  • specific numbering may be used for amino acid residue positions in proteins used in this application. For example, by aligning the polypeptide sequence of the protein of the present application with the target protein to be compared, it is possible to renumber the position corresponding to the amino acid residue position of the protein of the present application.
  • the glutamate-cysteine ligase variant is the amino acid sequence of SEQ ID NO: 1 or 80%, 85%, 90% thereof %, 95%, 96%, 97%, 98%, or a protein in which the amino acid corresponding to position 653 of SEQ ID NO: 1 is substituted with methionine among amino acid sequences having homology or identity of at least 99%.
  • Such a variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity with SEQ ID NO: 1, and less than 100% homology with SEQ ID NO: 1 Or it may be a variant having the same identity, but is not limited thereto.
  • the glutamate-cysteine ligase variant may include the amino acid sequence of SEQ ID NO: 3. Specifically, it may consist essentially of the amino acid sequence of SEQ ID NO: 3, and more specifically, it may consist of the amino acid sequence of any one of SEQ ID NO: 3, but is not limited thereto.
  • the variant includes the amino acid sequence of SEQ ID NO: 3, or amino acid 653 in the amino acid sequence is fixed (ie, the amino acid corresponding to position 653 of SEQ ID NO: 3 in the amino acid sequence of SEQ ID NO: 3 It is the same as the amino acid at position 653 of), and may include an amino acid sequence having 80% or more homology or identity therewith, but is not limited thereto.
  • the variant of the present application is a polypeptide having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity to SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 3 may include
  • a protein having an amino acid sequence in which some sequence is deleted, modified, substituted or added other than position 653 is also included in the scope of the present application. self-evident
  • the term 'homology' or 'identity' refers to a degree related to two given amino acid sequences or nucleotide sequences and may be expressed as a percentage.
  • the terms homology and identity can often be used interchangeably.
  • Sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, with default gap penalties established by the program used may be used.
  • Substantially, homologous or identical sequences generally have moderate or high stringency conditions along at least about 50%, 60%, 70%, 80% or 90% of the entire or full-length sequence. It can hybridize under stringent conditions. It is obvious that hybridization also includes polynucleotides containing common codons in polynucleotides or codons taking codon degeneracy into account.
  • a GAP program can be defined as the total number of symbols in the shorter of two sequences divided by the number of similarly aligned symbols (ie, nucleotides or amino acids).
  • Default parameters for the GAP program are: (1) a binary comparison matrix (containing values of 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation , pp. 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: weighted comparison matrix of 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for end gaps.
  • Glutamate-cysteine ligase variant in which the amino acid corresponding to position 653 from the N-terminus of the amino acid sequence of SEQ ID NO: 1 of the present application is substituted with methionine is additionally at position 86 from the N-terminus of the amino acid sequence of SEQ ID NO: 1 It may contain mutations in which the corresponding amino acid is substituted for another amino acid.
  • the variant may include a mutation in which the cysteine corresponding to position 86 is substituted with another amino acid, and may include, for example, a mutation in which arginine is substituted.
  • the variant may include, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 13. However, it is not limited thereto.
  • Another aspect of the present application may provide a polynucleotide encoding the variant.
  • polynucleotide refers to a DNA or RNA strand of a certain length or more as a polymer of nucleotides in which nucleotide monomers are connected in a long chain form by covalent bonds.
  • the gene encoding the glutamate-cysteine ligase of the present application may be a gsh1 gene.
  • the gene may be derived from yeast. Specifically, it may be derived from Saccharomyces genus, more specifically Saccharomyces cerevisiae. Specifically, glutamate derived from Saccharomyces cerevisiae includes without limitation as long as it encodes a polypeptide having cysteine ligase activity, and in one embodiment may be a gene encoding the amino acid sequence of SEQ ID NO: 1, one embodiment For example, it may include the nucleotide sequence of SEQ ID NO: 2, but is not limited thereto.
  • the polynucleotide encoding the protein variant of the present application may be included without limitation as long as it is a polynucleotide encoding a glutamate-cysteine ligase variant of the present application and a polypeptide having a corresponding activity.
  • the polynucleotide encoding the glutamate-cysteine ligase and variants thereof of the present application does not change the amino acid sequence of the polypeptide due to codon degeneracy or in consideration of codons preferred in the organism in which the polypeptide is to be expressed.
  • Various modifications may be made to the coding region within the non-limiting range.
  • the polynucleotide encoding the protein variant of the present application may include without limitation as long as it is a polynucleotide sequence encoding a protein variant in which the amino acid corresponding to position 653 in the amino acid sequence of SEQ ID NO: 1 is substituted with methionine.
  • the polynucleotide encoding the protein variant of the present application is a polynucleotide sequence encoding the protein variant of the present application, specifically, a protein comprising the amino acid sequence of SEQ ID NO: 3 or a polypeptide having homology or identity thereto may be, but is not limited thereto. The homology or identity is the same as described above.
  • polynucleotide encoding the protein variant of the present application is hybridized under stringent conditions with a probe that can be prepared from a known gene sequence, for example, a sequence complementary to all or part of the base sequence, SEQ ID NO: 1 Any sequence encoding a protein variant in which the amino acid corresponding to position 653 in the amino acid sequence of is substituted with methionine may be included without limitation.
  • stringent condition refers to a condition that enables specific hybridization between polynucleotides. These conditions are specifically described in the literature (eg, J. Sambrook et al., 1989, supra). For example, polynucleotides with high homology or identity are 40% or more, specifically 90% or more, more specifically 95% or more, 96% or more, 97% or more, 98% or more, and more specifically 99% or more.
  • Conditions in which polynucleotides having homology or identity hybridize with each other and polynucleotides with lower homology or identity do not hybridize or wash conditions of conventional Southern hybridization at 60° C., 1 ⁇ SSC, 0.1 at a salt concentration and temperature equivalent to % SDS, specifically 60° C., 0.1 ⁇ SSC, 0.1% SDS, more specifically 68° C., 0.1 ⁇ SSC, 0.1% SDS, washing once, specifically 2 to 3 times Conditions can be enumerated.
  • Hybridization requires that two nucleic acids have complementary sequences, although mismatch between bases is possible depending on the stringency of hybridization.
  • the term “complementary” is used to describe the relationship between nucleotide bases capable of hybridizing to each other. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the polynucleotides of the present application may also include substantially similar nucleic acid sequences as well as isolated nucleic acid fragments complementary to the overall sequence.
  • polynucleotides having homology or identity can be detected using hybridization conditions including a hybridization step at a Tm value of 55° C. and using the above-described conditions.
  • the Tm value may be 60 °C, 63 °C, or 65 °C, but is not limited thereto and may be appropriately adjusted by those skilled in the art according to the purpose.
  • Another aspect of the present application may provide a vector comprising a polynucleotide encoding the protein variant.
  • vector refers to a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so that the target polypeptide can be expressed in a suitable host.
  • the expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation.
  • the vector After transformation into an appropriate host cell, the vector can replicate or function independently of the host genome, and can be integrated into the genome itself.
  • a polynucleotide encoding a target protein in a chromosome may be replaced with a mutated polynucleotide through a vector for intracellular chromosome insertion. Insertion of the polynucleotide into a chromosome may be performed by any method known in the art, for example, homologous recombination, but is not limited thereto. It may further include a selection marker (selection marker) for confirming whether the chromosome is inserted.
  • the selection marker is used to select cells transformed with the vector, that is, to confirm whether a target nucleic acid molecule is inserted, and to confer a selectable phenotype such as drug resistance, auxotrophicity, resistance to cytotoxic agents, or surface protein expression. markers may be used. In an environment treated with a selective agent, only the cells expressing the selectable marker survive or exhibit other expression traits, so that the transformed cells can be selected.
  • the vector used in the present application is not particularly limited, and any vector known in the art may be used.
  • the yeast expression vector can be both an integrative yeast plasmid (YIp) and an extrachromosomal plasmid vector.
  • the extrachromosomal plasmid vector may include an episomal yeast plasmid (YEp), a replicative yeast plasmid (YRp), and a yeast centromer plasmid (YCp).
  • YEp episomal yeast plasmid
  • YRp replicative yeast plasmid
  • YCp yeast centromer plasmid
  • artificial yeast chromosomes YACs
  • available vectors are pESCHIS, pESC-LEU, pESC-TRP, pESC-URA, Gateway pYES-DEST52, pAO815, pGAPZ A, pGAPZ B, pGAPZ C, pGAPa A, pGAPa B, pGAPa C, pPIC3.5K , pPIC6 A, pPIC6 B, pPIC6 C, pPIC6 ⁇ A, pPIC6 ⁇ B, pPIC6 ⁇ C, pPIC9K, pYC2/CT, pYD1 Yeast Display Vector, pYES2, pYES2/CT, pYES2/NT A, pYES2/NT B, pYES2/NT C , pYES2/CT, pYES2.1, pYES-DEST52, pTEF1/Zeo, pFLD1, PichiaPinkTM, p4
  • the term “transformation” refers to introducing a vector including a polynucleotide encoding a target protein into a host cell or microorganism so that the protein encoded by the polynucleotide can be expressed in the host cell.
  • the transformed polynucleotide may include all of them regardless of whether they are inserted into the chromosome of the host cell or located extrachromosomally, as long as they can be expressed in the host cell.
  • the polynucleotide includes DNA and RNA encoding a target protein.
  • the polynucleotide may be introduced in any form as long as it can be introduced and expressed into a host cell.
  • the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct including all elements necessary for self-expression.
  • the expression cassette may include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal.
  • the expression cassette may be in the form of an expression vector capable of self-replication.
  • the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto.
  • operably linked means that a promoter sequence that initiates and mediates transcription of a polynucleotide encoding a target polypeptide of the present application and the gene sequence are functionally linked.
  • the method for transforming the vector of the present application includes any method of introducing a nucleic acid into a cell, and may be performed by selecting a suitable standard technique as known in the art depending on the host cell. For example, electroporation, calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and Lithium acetate-DMSO method and the like, but is not limited thereto.
  • the present application relates to the variant; a polynucleotide encoding the variant; And it may include any one or more of vectors including the polynucleotide, to provide a microorganism producing glutathione.
  • microorganism includes both wild-type microorganisms and microorganisms in which genetic modification has occurred, either naturally or artificially, and a specific mechanism is weakened due to causes such as insertion of an external gene or enhanced or weakened activity of an intrinsic gene. It is a concept that includes all microorganisms that have been or have been fortified.
  • the microorganism in the present application may be included without limitation as long as it is a microorganism into which the glutamate-cysteine ligase variant of the present application is introduced or included.
  • the microorganism is transformed with a gene encoding a target protein or a vector comprising the same, for example, as a cell or microorganism expressing the target protein, and for the purpose of the present application, the host cell or microorganism is the glutamate-cysteine ligase Any microorganism capable of producing glutathione, including mutants, is possible.
  • Glutathione is used interchangeably with “glutathione” and “GSH”, and refers to a tripeptide composed of three amino acids: glutamate, cysteine, and glycine. Glutathione may be used as a raw material for pharmaceuticals, health functional foods, flavoring materials, food, feed additives, cosmetics, etc., but is not limited thereto.
  • glutathione-producing microorganism includes all microorganisms in which genetic modification has occurred, either naturally or artificially, and a specific mechanism is weakened due to causes such as insertion of an external gene or intensification or inactivation of the activity of an intrinsic gene
  • the modified or enhanced microorganism it may be a microorganism having a genetic mutation or enhanced activity for the desired glutathione production.
  • the glutathione-producing microorganism may refer to a microorganism capable of producing a desired glutathione in excess compared to a wild-type or unmodified microorganism, including glutamate-cysteine ligase.
  • the “glutathione-producing microorganism” may be used interchangeably with terms such as “glutathione-producing microorganism”, “microorganism having glutathione-producing ability,” “glutathione-producing strain”, “strain having glutathione-producing ability”, and the like.
  • the glutathione-producing microorganism is not particularly limited in its type as long as glutathione production is possible, but may be a microorganism of the genus Saccharomyces , specifically Saccharomyces cerevisiae ). However, the present invention is not limited thereto.
  • the parent strain of the glutathione-producing microorganism including the mutant is not particularly limited as long as it is capable of producing glutathione.
  • the microorganism may further include mutations such as enhancement of biosynthetic pathways for increasing glutathione production capacity, release of feedback inhibition, gene inactivation that weakens degradation pathways or biosynthetic pathways, and such mutations do not exclude natural ones .
  • the microorganism may include a mutation in the expression control region of glutamate-cysteine ligase to increase glutathione production ability.
  • variants of the present application a polynucleotide encoding the variant; and the microorganism comprising any one or more of the vector containing the polynucleotide, glutamate-cysteine ligase variant in which the amino acid corresponding to position 653 in the amino acid sequence of SEQ ID NO: 1 is substituted with methionine may be a microorganism It is not limited thereto.
  • the glutamate-cysteine ligase and its variants are as described above.
  • the term "to be/are" a protein refers to a state in which a target protein is introduced into a microorganism or modified to be expressed in the microorganism.
  • the target protein is a protein present in a microorganism, it refers to a state in which the activity is enhanced compared to before intrinsic or modification.
  • the microorganism expressing the protein variant of the present application may be a microorganism modified to express the protein variant of the present application, and thus another aspect of the present application provides a method for producing a microorganism expressing the protein variant of the present application. .
  • introduction of protein refers to exhibiting the activity of a specific protein that the microorganism did not originally have, or exhibiting improved activity compared to the intrinsic activity or activity prior to modification of the corresponding protein.
  • a specific protein is introduced, a polynucleotide encoding a specific protein is introduced into a chromosome in a microorganism, or a vector including a polynucleotide encoding a specific protein is introduced into a microorganism to exhibit its activity.
  • the term “enhancement” of polypeptide or protein activity means that the activity of the polypeptide or protein is increased compared to the intrinsic activity.
  • the reinforcement may be used interchangeably with terms such as up-regulation, overexpression, and increase.
  • the increase may include both exhibiting an activity that it did not originally have, or exhibiting an improved activity compared to intrinsic activity or activity before modification.
  • intrinsic activity refers to the activity of a specific polypeptide or protein originally possessed by the parent strain or unmodified microorganism before the transformation when the trait is changed due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with “activity before modification”.
  • “Enhancement” or “increase” in the activity of a polypeptide or protein compared to its intrinsic activity means that it is improved compared to the activity of a specific polypeptide or protein originally possessed by the parent strain or unmodified microorganism before transformation.
  • the “increase in activity” may be achieved by introducing an exogenous polypeptide or protein or enhancing the activity of an endogenous polypeptide or protein, but specifically, it may be achieved through enhancing the activity of an endogenous polypeptide or protein. Whether the activity of the polypeptide or protein is enhanced can be confirmed from an increase in the activity level, expression level, or amount of a product excreted from the corresponding polypeptide or protein.
  • the enhancement of the activity of the polypeptide or protein can be applied by various methods well known in the art, and may not be limited as long as it can enhance the activity of the target polypeptide or protein compared to the microorganism before modification.
  • the method is not limited thereto, but may use genetic engineering and/or protein engineering well known to those skilled in the art, which are routine methods of molecular biology (Sitnicka et al . Functional Analysis of Genes. Advances in Cell Biology). 2010, Vol. 2. 1-16, Sambrook et al . Molecular Cloning 2012 et al.).
  • the method for enhancing polypeptide or protein activity using the protein engineering may be performed by, for example, a method of selecting an exposed site by analyzing the tertiary structure of the polypeptide or protein and modifying or chemically modifying it, but is limited thereto. doesn't happen
  • the increase in the intracellular copy number of a gene or polynucleotide encoding a polypeptide or protein is performed by any method known in the art, for example, the gene or polynucleotide encoding the polypeptide or protein is operably linked, This can be performed by introducing a vector capable of replicating and functioning independently of a host into a host cell. Alternatively, a vector capable of inserting the gene or polynucleotide into a chromosome in the host cell, to which the gene is operably linked, may be introduced into the host cell, but is not limited thereto. The vector is the same as described above.
  • the method of replacing the gene expression control region (or expression control sequence) on the chromosome encoding the polypeptide or protein with a sequence with strong activity is any method known in the art, for example, the activity of the expression control region It can be carried out by inducing a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution or a combination thereof to further enhance the nucleic acid sequence, or by replacing the nucleic acid sequence with a nucleic acid sequence having stronger activity.
  • the expression control region is not particularly limited thereto, but may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence for regulating the termination of transcription and translation. The method may specifically be to link a strong heterologous promoter instead of the original promoter, but is not limited thereto.
  • promoters known for eukaryotes include translation elongation factor 1 (TEF1), glycerol-3-phosphate dehydrogenase 1 (GPD1), 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase, glycer Aldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosphosphate
  • yeast promoters that are inducible promoters that may include promoters for isomerase, phosphoglucose isomerase, and glucokinase and have the added advantage of transcription controlled by growth conditions include alcohol dehydrogenase second, isocytochromium C, acid phosphatase, degradative enzymes involved in nitrogen metabolism, metallothionein,
  • the method of modifying the base sequence of the start codon or 5'-UTR region of the polypeptide or protein is any method known in the art, for example, the endogenous start codon of the polypeptide or protein is added to the endogenous start codon. It may be substituted with another start codon having a higher expression rate of a polypeptide or protein than that, but is not limited thereto.
  • the method of modifying a polynucleotide sequence on a chromosome to increase polypeptide or protein activity may include any method known in the art, for example, deleting, inserting, or inserting a nucleic acid sequence to further enhance the activity of the polynucleotide sequence; It can be carried out by inducing a mutation in the expression control sequence by non-conservative or conservative substitution or a combination thereof, or by replacing it with an improved polynucleotide sequence to have stronger activity. The replacement may specifically be to insert the gene into the chromosome by homologous recombination, but is not limited thereto.
  • the vector used may further include a selection marker for confirming whether or not the chromosome is inserted.
  • the selection marker is the same as described above.
  • a foreign polynucleotide exhibiting the activity of a polypeptide or protein can be carried out by any method known in the art, for example, a foreign polynucleotide encoding a polypeptide or protein exhibiting the same/similar activity as the polypeptide or protein; Or it may be carried out by introducing a codon-optimized variant polynucleotide thereof into a host cell.
  • the foreign polynucleotide may be used without limitation in origin or sequence as long as it exhibits the same/similar activity as the polypeptide or protein.
  • the introduced foreign polynucleotide can be introduced into the host cell by optimizing its codon so that the optimized transcription and translation are performed in the host cell.
  • the introduction can be carried out by appropriately selecting a known transformation method by those skilled in the art, and by expressing the introduced polynucleotide in a host cell, a polypeptide or protein is produced and its activity can be increased.
  • the combination of the methods may be performed by applying any one or more methods of 1) to 5) together.
  • the enhancement of such polypeptide or protein activity is a function in which the activity or concentration of the corresponding polypeptide or protein is increased relative to the activity or concentration of the polypeptide or protein expressed in the wild-type or pre-modified microbial strain, or is produced from the polypeptide or protein.
  • the amount of the product may be increased, but is not limited thereto.
  • pre-transformation strain or "pre-transformation microorganism” does not exclude a strain containing a mutation that can occur naturally in a microorganism, it is a wild-type strain or a natural-type strain itself, or caused by natural or artificial factors It may refer to a strain before the trait is changed due to genetic mutation.
  • pre-modified strain or “pre-modified microorganism” may be used interchangeably with “unmodified strain”, “unmodified strain”, “unmodified microorganism”, “unmodified microorganism” or “reference microorganism”.
  • a microorganism comprising a polynucleotide comprising or encoding the glutamate-cysteine ligase variant, or a vector comprising the polynucleotide may be a recombinant microorganism, and the recombination is genetic modification such as transformation ( genetically modified).
  • the recombinant microorganism may be a recombinant microorganism prepared by transformation with a vector containing the polynucleotide, but is not limited thereto.
  • the recombinant microorganism may be yeast, for example, may be a microorganism of the genus Saccharomyces, specifically Saccharomyces cerevisiae , but may be, but is not limited thereto.
  • Another aspect of the present application provides a glutathione production method comprising the step of culturing the microorganism.
  • the microorganism, glutathione, is the same as described above.
  • any medium and other culture conditions used for culturing the strain of the present application may be used without any particular limitation as long as it is a medium used for culturing a microorganism of the genus Saccharomyces, specifically, the strain of the present application is selected from a suitable carbon source; It can be cultured while controlling temperature, pH, etc. under aerobic or anaerobic conditions in a normal medium containing a nitrogen source, phosphorus, inorganic compounds, amino acids and/or vitamins.
  • carbohydrates such as glucose, fructose, sucrose, maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, citric acid and the like; Amino acids such as glutamate, methionine, lysine and the like may be included, but are not limited thereto.
  • natural organic nutrient sources such as starch hydrolyzate, molasses, blackstrap molasses, rice winter, cassava, sugarcane offal and corn steep liquor can be used, including glucose and pasteurized pretreated molasses (i.e. molasses converted to reducing sugar).
  • Carbohydrates such as, etc. may be used, and other appropriate amounts of carbon sources may be variously used without limitation. These carbon sources may be used alone or in combination of two or more.
  • nitrogen source examples include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Organic nitrogen sources such as amino acids, peptones, NZ-amines, meat extracts, yeast extracts, malt extracts, corn steep liquor, casein hydrolysates, fish or degradation products thereof, defatted soybean cakes or degradation products thereof and the like can be used. These nitrogen sources may be used alone or in combination of two or more, but is not limited thereto.
  • inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate
  • Organic nitrogen sources such as amino acids, peptones, NZ-amines, meat extracts, yeast extracts, malt extracts, corn steep liquor, casein hydrolysates, fish or degradation products thereof, de
  • the phosphorus may include potassium monobasic phosphate, dipotassium phosphate, or a sodium-containing salt corresponding thereto.
  • potassium monobasic phosphate dipotassium phosphate
  • sodium-containing salt corresponding thereto.
  • sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. may be used.
  • the medium may contain amino acids, vitamins and/or appropriate precursors.
  • L-amino acid and the like may be added to the culture medium of the strain.
  • glycine, glutamate, and/or cysteine may be added, and if necessary, L-amino acids such as lysine may be further added, but not necessarily limited thereto. does not
  • the medium or precursor may be added to the culture in a batch or continuous manner, but is not limited thereto.
  • the pH of the culture can be adjusted.
  • an antifoaming agent such as fatty acid polyglycol ester may be used to suppress bubble formation.
  • oxygen or oxygen-containing gas may be injected into the culture, or nitrogen, hydrogen or carbon dioxide gas may be injected with or without gas to maintain anaerobic and microaerobic conditions.
  • the temperature of the culture may be 25°C to 40°C, and more specifically, 28°C to 37°C, but is not limited thereto.
  • the incubation period may be continued until a desired production amount of useful substances is obtained, and specifically, it may be 1 hour to 100 hours, but is not limited thereto.
  • the glutathione production method may further include an additional process after the culturing step.
  • the additional process may be appropriately selected depending on the use of glutathione.
  • the glutathione production method may include the step of recovering glutathione accumulated in the cells by the culturing step, for example, after the culturing step, one selected from the strain, its dried product, extract, culture, and lysate. It may include the step of recovering glutathione from the above substances.
  • the method may further include a step of lysing the strain before or simultaneously with the recovery step.
  • the lysis of the strain may be carried out by a method commonly used in the technical field to which the present application belongs, for example, a lysis buffer solution, a sonicator, heat treatment, and a French presser.
  • the lysis step may include an enzymatic reaction such as a cell wall degrading enzyme, a nucleic acid degrading enzyme, a nucleic acid transfer enzyme, a proteolytic enzyme, but is not limited thereto.
  • dry yeast containing a high content of glutathione through the glutathione manufacturing method yeast extract, yeast extract mix powder, pure purified glutathione (pure) glutathione), but is not limited thereto, and may be appropriately manufactured according to the desired product.
  • dry yeast may be used interchangeably with terms such as "dried strain”.
  • the dry yeast may be prepared by drying yeast cells accumulating glutathione, and may be specifically included in a composition for feed, a composition for food, and the like, but is not limited thereto.
  • yeast extract may be used interchangeably with terms such as "strain extract".
  • the strain extract may mean a material remaining after separating the cell wall from the cells of the strain. Specifically, it may refer to the remaining components excluding the cell wall from the components obtained by lysing the cells.
  • the extract of the strain includes glutathione, and components other than glutathione may include at least one of protein, carbohydrate, nucleic acid, and fiber, but is not limited thereto.
  • glutathione a target material
  • glutathione a target material
  • the recovery step may include a purification process.
  • the purification process may be pure purification by separating only glutathione from the strain. Through the purification process, pure purified glutathione may be prepared.
  • the method for preparing glutathione may further include a step of mixing a material selected from among the strains obtained after the culturing step, a dried product, an extract, a culture, a lysate, and glutathione recovered therefrom and an excipient.
  • yeast extract mix powder can be prepared.
  • the excipient may be appropriately selected and used according to the intended use or form, for example, starch, glucose, cellulose, lactose, glycogen, D-mannitol, sorbitol, lactitol, maltodextrin, calcium carbonate, synthetic aluminum silicate, Calcium monohydrogen phosphate, calcium sulfate, sodium chloride, sodium hydrogen carbonate, purified lanolin, dextrin, sodium alginate, methylcellulose, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethylcellulose, propylene glycol, casein, calcium lactate , Primogel, and gum arabic, specifically, starch, glucose, cellulose, lactose, dextrin, glycogen, D-mannitol, may be one or more components selected from maltodextrin, but is not limited thereto.
  • the excipient may include, for example, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizing agent, or an isotonic agent, but is not limited thereto.
  • Another aspect of the present application provides a glutathione production use of the variant of the present application.
  • One aspect of the present application provides a glutathione production use of a microorganism comprising the mutant glutamate-cysteine ligase variant of the present application.
  • Example 1-1 Selection of glutathione-producing strains
  • a strain having a glutathione-producing ability was selected by obtaining a strain from yeast containing various strains and improving it.
  • grain samples such as rice, barley, mung bean, and oats are collected from a total of 20 regions including Yongin, Icheon, Pyeongtaek, and Hwaseong areas in Gyeonggi-do, Korea, and then kneaded, wrapped in a cloth, pressed firmly to form a shape, and then wrapped with straw and fermented for 10 days. After that, it was dried slowly to prepare yeast.
  • 25ml of YPD broth was dispensed in a 250ml Erlenmeyer flask, inoculated with the pure isolated strain, and cultured with shaking (30°C, 200rpm) for 48 hours to check the glutathione production, and strain screening was performed.
  • a mutation (random mutation) was induced in the isolated strain. Specifically, a strain in which glutathione production was confirmed among the yeast isolated from the yeast was isolated and named CJ-37 strain. After culturing the CJ-37 strain on a solid medium, the broth was inoculated to obtain a culture solution, and UV was irradiated to the cells using a UV lamp. Thereafter, only mutant strains that formed colonies were isolated and obtained by smearing the UV-irradiated culture medium on a plate medium, and their glutathione production was confirmed.
  • the strain showing the best glutathione production among the mutated strains was selected as the glutathione-producing strain and named CJ-5 strain. It was deposited on the 31st of the month and given the deposit number KCCM12568P.
  • Example 1-2 Additional improvement experiment for increasing glutathione production capacity
  • Example 1-3 Additional improvement experiment for increasing glutathione production capacity
  • the broth was inoculated to obtain a culture solution, and UV was irradiated to the cells using a UV lamp. After that, only the mutant strains that formed colonies were isolated and obtained by smearing the UV-irradiated culture medium on a plate medium, and the strain with the most improved glutathione production ability was isolated and named as the CC02-2544 strain. It was deposited with the (Korean Culture Center of Microorganisms, KCCM) on February 20, 2020 and was given an accession number KCCM12674P.
  • Example 2 Additional improvement experiment of strain CC02-2544 for increasing glutathione production capacity
  • the broth was inoculated to obtain a culture solution, and UV was irradiated to the cells using a UV lamp. Thereafter, only mutant strains that formed colonies by spreading the UV-irradiated culture medium on a plate medium were isolated and obtained and the base sequence was analyzed.
  • Example 2 From the results of Example 2, it was determined that the 653 position of the GSH1 protein would be important for glutathione production, and S. cerevisiae (S. cerevisiae) CEN.PK2-1D and CC02-2544 strains were prepared to determine whether glutathione production was increased. Meanwhile, as mentioned above, in the case of the CC02-2544 strain, the GSH1 ORF upper end -250(C ⁇ T), -252(G ⁇ A), -398(A ⁇ T), -399(A ⁇ C), -407 (T ⁇ C), -409 (T ⁇ C) is a strain with mutations, and a mutation of amino acid 653 of the GSH1 protein was additionally introduced into the strain.
  • PCR was performed using the primers of SEQ ID NO: 4 and SEQ ID NO: 5 to secure a partial sequence of GSH1 N-terminal including the N-terminal BamHI flanking sequence, the start codon of GSH1 ORF, and the G653M variant coding sequence, and SEQ ID NO: 6 and the sequence
  • a partial sequence of GSH1 C-terminal including the C-terminal XhoI flanking sequence, the GSH1 ORF stop codon and the G653M mutation coding sequence was obtained by using the primer of No. 7.
  • PCR using SEQ ID NO: 8 and SEQ ID NO: 9 using the genomic DNA of the CJ-5 strain as a template was performed to secure a 500 bp fragment after the GSH1 ORF stop codon including the N-terminal SpeI and C-terminal NcoI restriction enzyme sequences and SpeI and NcoI restriction enzymes were treated.
  • pWBR100-GSH1 vector was prepared.
  • each PCR product was obtained S. cerevisiae CEN.PK2-1D and S. cerevisiae CC02-2544 were transformed at the same molar ratio.
  • PCR was performed under the conditions of 5 minutes for heat denaturation at 95°C, 1 minute for binding at 53°C, and 1 minute for polymerization at 72°C for 1 minute per kb. (6), 1425) modified lithium acetate method (Lithium acetate method) was used. Specifically, O.D. After washing yeast cells between 0.7 and 1.2 twice with lithium acetate/TE buffer, the PCR products and single stranded DNA (Sigma D-7656) were mixed together in lithium acetate ate/TE/40% PEG buffer for 30 minutes.
  • a strain capable of expressing the GSH1 mutant protein substituted with an amino acid other than methionine also uses a primer pair in which the methionine coding sequence #653 on the primer sequences of SEQ ID NOs: 5 and 6 is substituted with a sequence encoding another amino acid. It was manufactured in the same way except for
  • GSH glutathione
  • Tables 2 and 3 The results of measuring the glutathione (GSH) concentration and content produced by culturing each strain prepared above for 26 hours are shown in Tables 2 and 3. Additional introduction of GSH1 G653M mutation into the CC02-2544 strain (SEQ ID NO: 13) As a result, the GSH concentration increased by 77 mg/L from 471.5 mg/L to 548.5 mg/L, and as a result of introducing the GSH1 G653M mutation (SEQ ID NO: 3) into the CEN.PK-1D strain, the GSH concentration increased from 42 mg/L to 100 mg/L increased by 58 mg/L.
  • Example 3-1 GSH1 G653 mutation introduced into the CC02-2544 strain
  • Example 3-2 GSH1 G653 mutation introduced into CEN.PK-1D strain
  • the novel GSH1 variant developed in the present application indicates an increase in glutathione production.
  • the yeast that produces high glutathione, including the GSH1 mutant of the present application, its dried product, extract, culture, lysate, and produced glutathione have antioxidant effects, detoxification effects, and immunity enhancing effects, cosmetic compositions, It can be usefully used in a composition for food, a composition for feed, a pharmaceutical composition, and the preparation thereof.
  • PCR was performed using the primers of SEQ ID NO: 4 and SEQ ID NO: 14 to secure a partial sequence of GSH1 N-terminal including the N-terminal BamHI flanking sequence, the initiation codon of the GSH1 ORF and the C86R variant coding sequence, and SEQ ID NO: 15 and the sequence
  • a partial sequence of GSH1 C-terminal including the C-terminal XhoI flanking sequence, the GSH1 ORF stop codon and the C86R mutation coding sequence was obtained using the primer of No. 7.
  • each PCR product was obtained S. cerevisiae CEN.PK2-1D and S. cerevisiae CJ-5 were transformed at the same molar ratio.
  • PCR was carried out under the conditions of 5 minutes for heat denaturation at 95°C, 1 minute for binding at 53°C, and 1 minute for polymerization at 72°C for 1 minute per kb. , 1425), a modified lithium acetate method was used. Specifically, O.D.
  • a strain capable of expressing the GSH1 mutant protein substituted with other amino acids other than arginine also uses a primer pair in which the arginine coding sequence 86 on the primer sequences of SEQ ID NOs: 14 and 15 is substituted with a sequence encoding another amino acid. and produced in the same way.

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Abstract

The present application relates to a novel glutamate-cysteine ligase variant and a method for producing glutathione using same.

Description

글루타메이트-시스테인 리가아제 변이체 및 이를 이용한 글루타치온 생산방법Glutamate-cysteine ligase variant and glutathione production method using the same
본 출원은 신규한 글루타메이트-시스테인 리가아제 변이체 및 이를 이용한 글루타치온 생산방법에 관한 것이다.The present application relates to a novel glutamate-cysteine ligase variant and a glutathione production method using the same.
글루타치온(Glutathione, GSH)은 세포 내에서 가장 일반적으로 존재하는 유기 황화합물로 글리신(glycine), 글루타메이트(glutamate), 시스테인(cysteine)의 세 가지 아미노산들이 결합한 트리펩타이드(tripeptide) 형태이다. Glutathione (GSH) is an organic sulfur compound most commonly present in cells, and is in the form of a tripeptide in which three amino acids of glycine, glutamate, and cysteine are combined.
글루타치온은 체내에서는 환원형 글루타치온(GSH)과 산화형 글루타치온(GSSG) 두 가지 형태로 존재한다. 일반적인 상황에서 상대적으로 높은 비율로 존재하는 환원형 글루타치온(GSH)은 인체의 간과 피부 세포에 주로 분포하며 활성산소를 분해·제거하는 항산화 기능, 독성물질과 같은 외인성 화합물의 제거 등 해독 작용, 멜라닌 색소 생성을 억제하는 미백작용 등 중요한 역할을 한다.Glutathione exists in the body in two forms: reduced glutathione (GSH) and oxidized glutathione (GSSG). Reduced glutathione (GSH), which is present in a relatively high proportion under normal circumstances, is mainly distributed in the liver and skin cells of the human body. It plays an important role, such as a whitening action that inhibits the production.
노화가 진행될수록 글루타치온 생성량은 점점 감소하며, 항산화, 해독작용에 중요한 역할을 하는 글루타치온 생성량의 감소는 노화의 주범인 활성산소의 축적을 촉진시키므로, 외부에서 글루타치온의 공급이 필요하다(Sipes IG et al, The role of glutathione in the toxicity of xenobiotic compounds: metabolic activation of 1,2-dibromoethane by glutathione, Adv Exp Med Biol. 1986;197:457-67.).As aging progresses, the amount of glutathione produced gradually decreases, and the decrease in the production of glutathione, which plays an important role in antioxidant and detoxification, promotes the accumulation of active oxygen, the main culprit of aging, so glutathione supply is required from the outside (Sipes IG et al. , The role of glutathione in the toxicity of xenobiotic compounds: metabolic activation of 1,2-dibromoethane by glutathione, Adv Exp Med Biol. 1986;197:457-67.).
이와 같이 다양한 기능을 지닌 글루타치온은 제약, 건강기능식품, 화장품 등 다양한 분야의 소재로 각광받고 있으며 맛소재, 식품 및 사료 첨가제 제조에 이용되기도 한다. 글루타치온은 원물의 맛 상승과 풍부한 맛 지속성 유지효과가 크며, 단독으로 사용하거나 다른 물질과 배합하여 고쿠미(kokumi) 향미증진제로 사용 할 수 있음이 알려져 있다. 보통 고쿠미 소재는 기존 핵산, MSG 등의 우마미(umami) 소재보다 농후감을 가지며, 단백질이 분해 숙성되어 생성되는 것으로 알려져 있다.As such, glutathione with various functions has been spotlighted as a material in various fields such as pharmaceuticals, health functional foods, and cosmetics, and is also used in the manufacture of flavor materials, food and feed additives. It is known that glutathione has a great effect of increasing the taste of the raw material and maintaining rich taste, and can be used alone or in combination with other substances as a kokumi flavor enhancer. Usually, Kokumi material has a richer feeling than umami material such as nucleic acid and MSG, and is known to be produced by decomposition and aging of protein.
그러나 이와 같이 다양한 분야에 사용할 수 있는 글루타치온에 대한 수요가 증가되고 있음에도 불구하고, 높은 생산 단가로 인해 효소 합성 공정은 아직 상용화가 되어 있지 않아, 글루타치온의 산업적 생산에 적지 않은 비용이 필요하기 때문에 시장이 크게 활성화되지 못하는 실정이다.However, despite the increasing demand for glutathione that can be used in various fields, the enzyme synthesis process has not yet been commercialized due to the high production cost. It is not very active.
본 출원자들은 새롭게 개발한 글루타메이트-시스테인 리가아제 변이체를 도입한 미생물이 글루타치온을 고수율로 제조할 수 있음을 확인하고, 본 출원을 완성하였다. The present applicants confirmed that a microorganism introducing a newly developed glutamate-cysteine ligase variant can produce glutathione in high yield, and completed the present application.
본 출원은 글루타메이트-시스테인 리가아제 활성을 갖는 단백질에서 서열번호 1의 아미노산 서열의 N-말단으로부터 653째 위치에 상응하는 아미노산이 메티오닌으로 치환된 글루타메이트-시스테인 리가아제 변이체를 제공한다.The present application provides a glutamate-cysteine ligase variant in which the amino acid corresponding to position 653 from the N-terminus of the amino acid sequence of SEQ ID NO: 1 in a protein having glutamate-cysteine ligase activity is substituted with methionine.
본 출원은 상기 변이체를 코딩하는 폴리뉴클레오티드, 및 이를 포함하는 벡터를 제공한다.The present application provides a polynucleotide encoding the variant, and a vector comprising the same.
본 출원은 상기 변이체; 상기 변이체를 코딩하는 폴리뉴클레오티드; 및 상기 폴리뉴클레오티드를 포함하는 벡터 중 어느 하나 이상을 포함하여 글루타치온을 생산하는 미생물을 제공한다. The present application relates to the variant; a polynucleotide encoding the variant; And it provides a microorganism for producing glutathione, including any one or more of the vector containing the polynucleotide.
본 출원은 상기 미생물을 배양하는 단계를 포함하는 글루타치온 생산 방법을 제공한다.The present application provides a glutathione production method comprising the step of culturing the microorganism.
본 출원의 신규한 글루타메이트-시스테인 리가아제 변이체는 글루타치온 생산을 크게 증가시키므로 글루타치온의 고생산에 유용하게 이용될 수 있는바, 이와 같이 글루타치온을 고생산하는 효모, 이의 건조물, 추출물, 배양물, 파쇄물 및 생산된 글루타치온은, 항산화 효과, 해독 효과, 면역력 증강 효과를 가지는 것인바, 화장품용 조성물, 식품용 조성물, 사료용 조성물, 의약품 조성물 및 이의 제조에도 유용하게 사용될 수 있다.Since the novel glutamate-cysteine ligase variant of the present application greatly increases glutathione production, it can be usefully used for high glutathione production. The produced glutathione has an antioxidant effect, a detoxification effect, and an immune enhancing effect, and thus can be usefully used in cosmetic compositions, food compositions, feed compositions, pharmaceutical compositions, and preparation thereof.
이를 구체적으로 설명하면 다음과 같다. 한편, 본 출원에서 개시된 각각의 설명 및 실시형태는 각각의 다른 설명 및 실시 형태에도 적용될 수 있다. 즉, 본 출원에서 개시된 다양한 요소들의 모든 조합이 본 출원의 범주에 속한다. 또한, 하기 기술된 구체적인 서술에 의하여 본 출원의 범주가 제한된다고 볼 수 없다.This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present application fall within the scope of the present application. In addition, it cannot be seen that the scope of the present application is limited by the detailed description described below.
또한, 당해 기술분야의 통상의 지식을 가진 자는 통상의 실험만을 사용하여 본 출원에 기재된 본 출원의 특정 양태에 대한 다수의 등가물을 인지하거나 확인할 수 있다. 또한, 이러한 등가물은 본 출원에 포함되는 것으로 의도된다.In addition, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present application described herein. Also, such equivalents are intended to be covered by this application.
본 출원의 하나의 양태는, 글루타메이트-시스테인 리가아제 활성을 갖는 단백질에서 아미노산 치환을 포함하고, 상기 치환은 서열번호 1의 N-말단으로부터 653번째 위치에 상응하는 아미노산이 메티오닌으로 치환된 것을 포함하는, 글루타메이트-시스테인 리가아제(glutamate-cysteine ligase) 변이체를 제공할 수 있다. One aspect of the present application includes an amino acid substitution in a protein having glutamate-cysteine ligase activity, wherein the substitution is an amino acid corresponding to the 653th position from the N-terminus of SEQ ID NO: 1 Including that methionine is substituted , glutamate-cysteine ligase (glutamate-cysteine ligase) variants may be provided.
상기 변이체는 서열번호 1의 글루타메이트-시스테인 리가아제 아미노산 서열에서 N-말단으로부터 653번째 위치에 상응하는 아미노산인 글리신이 메티오닌으로 치환된 단백질 변이체일 수 있다. The variant may be a protein variant in which glycine, an amino acid corresponding to position 653 from the N-terminus in the glutamate-cysteine ligase amino acid sequence of SEQ ID NO: 1 is substituted with methionine.
본 출원의 "글루타메이트-시스테인 리가아제(glutamate-cysteine ligase, GCL)"는 "글루타메이트-시스테인 연결효소" 또는 "감마-글루타밀시스테인 신테타아제(gamma-glutamylcysteine synthetase, GCS)" 라고도 칭해지는 효소이다. 글루타메이트-시스테인 리가아제는 다음의 반응을 촉매하는 것으로 알려져 있다:"Glutamate-cysteine ligase (GCL)" of the present application is an enzyme also called "glutamate-cysteine ligase" or "gamma-glutamylcysteine synthetase (GCS)" . Glutamate-cysteine ligase is known to catalyze the following reactions:
L-glutamate + L-cysteine + ATP ↔ gamma-glutamyl cysteine + ADP + PiL-glutamate + L-cysteine + ATP ↔ gamma-glutamyl cysteine + ADP + Pi
또한 상기 글루타메이트-시스테인 리가아제가 촉매하는 반응은 글루타치온 합성의 첫 번째 단계인 것으로 알려져 있다.In addition, it is known that the reaction catalyzed by the glutamate-cysteine ligase is the first step in glutathione synthesis.
본 출원에서 글루타메이트-시스테인 리가아제의 아미노산 서열은 gsh1 유전자에 의해 코딩되는 아미노산 서열로, "GSH1 단백질"또는 "글루타메이트-시스테인 리가아제"로 지칭할 수 있다. 본 출원의 글루타메이트-시스테인 리가아제를 구성하는 아미노산 서열은 공지의 데이터 베이스인 NCBI의 GenBank에서 그 서열을 얻을 수 있다. 상기 글루타메이트-시스테인 리가아제는 서열번호 1의 아미노산 서열을 포함하거나, 이를 필수적으로 구성되거나(consisting essentially of), 이로 구성되는 단백질일 수 있으나, 이에 제한되지 않는다. 또 다른 일 예로, 상기 글루타메이트-시스테인 리가아제는 사카로마이세스 세레비지애(Saccharomyces cerevisiae) 유래일 수 있고, 다른 예로, 사카로마이세스 서열번호 1의 아미노산 서열에서 653번 위치에 상응하는 아미노산이 글리신인 것일 수 있다. 그러나 이에 제한되지 않으며 상기 아미노산 서열과 동일한 글루타메이트-시스테인 리가아제 활성을 갖는 서열은 제한 없이 포함될 수 있다. In the present application, the amino acid sequence of glutamate-cysteine ligase is an amino acid sequence encoded by the gsh1 gene, and may be referred to as "GSH1 protein" or "glutamate-cysteine ligase". The amino acid sequence constituting the glutamate-cysteine ligase of the present application can be obtained from a known database, GenBank of NCBI. The glutamate-cysteine ligase may be a protein comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 1, but is not limited thereto. In another example, the glutamate-cysteine ligase may be derived from Saccharomyces cerevisiae , and in another example, the amino acid corresponding to position 653 in the amino acid sequence of Saccharomyces SEQ ID NO: 1 is It may be glycine. However, the present invention is not limited thereto, and a sequence having the same glutamate-cysteine ligase activity as the amino acid sequence may be included without limitation.
구체적인 예를 들어, 본 출원의 글루타메이트-시스테인 리가아제는 서열번호 1의 아미노산 서열 또는 이와 80%, 85%, 90%, 95%, 96%, 97%, 98%, 또는 99% 이상의 상동성 또는 동일성을 갖는 아미노산 서열을 포함하는 단백질일 수 있다. 또한, 이러한 상동성 또는 동일성을 가지며 상기 단백질에 상응하는 효능을 나타내는 아미노산 서열이라면, 일부 서열이 결실, 변형, 치환 또는 부가된 아미노산 서열을 갖는 단백질도 본 출원의 변이 대상이 되는 단백질의 범위 내에 포함됨은 자명하다.As a specific example, the glutamate-cysteine ligase of the present application has the amino acid sequence of SEQ ID NO: 1 or at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology thereto, or It may be a protein comprising an amino acid sequence with identity. In addition, as long as it is an amino acid sequence having such homology or identity and exhibiting efficacy corresponding to the protein, a protein having an amino acid sequence in which some sequence is deleted, modified, substituted or added is also included within the scope of the protein subject to mutation of the present application. is self-evident
또한, 본 출원에서 글루타메이트-시스테인 리가아제의 일 예로, 서열번호 1의 아미노산 서열을 포함하는 단백질이라고 정의하였으나, 서열번호 1의 아미노산 서열 앞뒤로의 무의미한 서열 또는 자연적으로 발생할 수 있는 돌연변이, 혹은 이의 잠재성 돌연변이(silent mutation)를 제외하는 것이 아니며, 서열번호 1의 아미노산 서열로 이루어진 단백질과 서로 동일 또는 상응하는 활성을 가지는 경우라면 본 출원의 글루타메이트-시스테인 리가아제에 해당됨은 당업자에게 자명하다. In addition, in the present application, as an example of glutamate-cysteine ligase, it is defined as a protein including the amino acid sequence of SEQ ID NO: 1, but a meaningless sequence before and after the amino acid sequence of SEQ ID NO: 1 or a naturally occurring mutation, or its potential It is apparent to those skilled in the art that the glutamate-cysteine ligase of the present application corresponds to the case where the mutation (silent mutation) is not excluded, and if it has the same or corresponding activity as the protein consisting of the amino acid sequence of SEQ ID NO: 1.
즉, 본 출원에서 '특정 서열번호로 기재된 아미노산 서열을 갖는 단백질 또는 폴리펩티드', '특정 서열번호로 기재된 아미노산 서열을 포함하는 단백질 또는 폴리펩티드'라고 기재되어 있다 하더라도, 해당 서열번호의 아미노산 서열로 이루어진 폴리펩티드와 동일 혹은 상응하는 활성을 가지는 경우라면, 일부 서열이 결실, 변형, 치환 또는 부가된 아미노산 서열을 갖는 단백질도 본 출원에서 사용될 수 있음은 자명하다. That is, even if it is described as 'a protein or polypeptide having an amino acid sequence set forth in a specific SEQ ID NO:' or 'a protein or polypeptide comprising an amino acid sequence set forth in a specific SEQ ID NO:' in the present application, a polypeptide consisting of the amino acid sequence of the corresponding SEQ ID NO: It is apparent that a protein having an amino acid sequence in which some sequences have been deleted, modified, substituted or added may also be used in the present application, provided that it has the same or corresponding activity to .
본 출원에서 용어, "변이체(variant)" 또는 “변이형 폴리펩티드(modified polypeptide)”는 하나 이상의 아미노산이 보존적 치환(conservative substitution) 및/또는 변형(modification)에 있어서 상기 열거된 서열(the recited sequence)과 상이하나, 상기 단백질의 기능(functions) 또는 특성(properties)이 유지되는 단백질을 지칭한다. 본 출원의 목적상, 상기 변이체는 상기에서 설명한 글루타메이트-시스테인 리가아제 중 서열번호 1의 N-말단으로부터 653번째 위치에 상응하는 아미노산이 메티오닌으로 치환된 글루타메이트-시스테인 리가아제 변이체 또는 글루타메이트-시스테인 리가아제 활성을 갖는 변이형 폴리펩티드일 수 있다. 본 출원의 변이체는 "글루타메이트-시스테인 리가아제 변이체" 는 "글루타메이트-시스테인 리가아제 활성을 갖는 (변이형) 폴리펩티드", "GSH1 변이체"로도 기재될 수 있다.As used herein, the term “variant” or “modified polypeptide” refers to the recited sequence in which one or more amino acids are conservatively substituted and/or modified. ), but refers to a protein in which the functions or properties of the protein are maintained. For the purpose of the present application, the variant is a glutamate-cysteine ligase variant or glutamate-cysteine ligase in which the amino acid corresponding to the 653 position from the N-terminus of SEQ ID NO: 1 among the above-described glutamate-cysteine ligases is substituted with methionine It may be a variant polypeptide having activity. As for the variants of the present application, "glutamate-cysteine ligase variant" may also be described as "(mutant) polypeptide having glutamate-cysteine ligase activity" and "GSH1 variant".
상기 변이체는 수 개의 아미노산 치환, 결실 또는 부가에 의해 식별되는 서열(identified sequence)과 상이하다. 이러한 변이체는 일반적으로 상기 단백질의 아미노산 서열 중 하나 이상의 아미노산을 변형하고, 상기 변형된 단백질의 특성을 평가하여 식별될 수 있다. 즉, 변이체의 능력은 본래 단백질(native protein)에 비하여 증가되거나, 변하지 않거나, 또는 감소될 수 있다. 또한, 일부 변이체는 N-말단 리더 서열 또는 막전이 도메인(transmembrane domain)과 같은 하나 이상의 부분이 제거된 변이형 폴리펩티드를 포함할 수 있다. 다른 변이체는 성숙 단백질 (mature protein) 의 N- 및/또는 C-말단으로부터 일부분이 제거된 변이체를 포함할 수 있다. 상기 용어 “변이체” 또는 “변이형 폴리펩티드”는 변이형, 변형, 변이된 단백질, 변이 등의 용어(영문 표현으로는 modification, modified protein, mutant, mutein, divergent, variant 등)가 혼용되어 사용될 수 있으며, 변이된 의미로 사용되는 용어라면 이에 제한되지 않는다.The variant differs from the identified sequence by several amino acid substitutions, deletions or additions. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the protein and evaluating the properties of the modified protein. That is, the ability of the variant may be increased, unchanged, or decreased compared to the native protein. In addition, some variants may include variant polypeptides in which one or more portions, such as an N-terminal leader sequence or a transmembrane domain, have been removed. Other variants may include variants in which a portion is removed from the N- and/or C-terminus of the mature protein. The term "variant" or "mutant polypeptide" may be used interchangeably with terms such as variant, modified, mutated protein, mutant (in English, modification, modified protein, mutant, mutein, divergent, variant, etc.) , as long as it is a term used in a mutated meaning, it is not limited thereto.
본 출원의 목적상, 상기 변이체는 천연의 야생형 또는 비변형 단백질 대비 변이된 단백질의 활성이 증가되거나, 변이 전의 단백질, 천연의 야생형 폴리펩티드 또는 비변형 폴리펩티드에 비해 글루타치온 생산량을 증가시키는 것일 수 있으나, 이에 제한되지 않는다.For the purposes of the present application, the mutant increases the activity of the mutated protein compared to the native wild-type or unmodified protein, or increases the glutathione production compared to the protein before mutation, the natural wild-type polypeptide or the unmodified polypeptide. not limited
본 출원에서 용어 “보존적 치환(conservative substitution)”은 한 아미노산을 유사한 구조적 및/또는 화학적 성질을 갖는 또 다른 아미노산으로 치환시키는 것을 의미한다. 상기 변이체는 하나 이상의 생물학적 활성을 여전히 보유하면서, 예를 들어 하나 이상의 보존적 치환을 가질 수 있다. 이러한 아미노산 치환은 일반적으로 잔기의 극성, 전하, 용해도, 소수성, 친수성 및/또는 양친매성(amphipathic nature)에서의 유사성에 근거하여 발생할 수 있다. As used herein, the term “conservative substitution” refers to substituting one amino acid with another amino acid having similar structural and/or chemical properties. Such variants may have, for example, one or more conservative substitutions while still retaining one or more biological activities. Such amino acid substitutions may generally occur based on similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of the residues.
예를 들면, 전하를 띠는 곁사슬(electrically charged amino acid)을 갖는 아미노산 중 양으로 하전된 (염기성) 아미노산은 아르기닌, 라이신, 및 히스티딘을, 음으로 하전된 (산성) 아미노산은 글루탐산 및 아스파르트산을 포함하고; 전하를 띠지 않는 곁사슬(uncharged side chain)을 갖는 아미노산은 글리신, 알라닌, 발린, 류신, 이소류신, 메티오닌, 페닐알라닌, 트립토판, 프롤린, 세린, 쓰레오닌, 시스테인, 타이로신, 아스파라긴, 글루타민을 포함하는 것으로 분류할 수 있다.For example, among amino acids with electrically charged amino acids, positively charged (basic) amino acids are arginine, lysine, and histidine, and negatively charged (acidic) amino acids are glutamic acid and aspartic acid. including; Amino acids with uncharged side chains are classified as including glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. can do.
또한, 변이체는 폴리펩티드의 특성과 2차 구조에 최소한의 영향을 갖는 아미노산들의 결실 또는 부가를 포함할 수 있다. 예를 들면 폴리펩티드는 번역-동시에(co-translationally) 또는 번역-후에(post-translationally) 단백질의 이전(transfer)에 관여하는 단백질 N-말단의 시그널(또는 리더) 서열과 컨쥬게이트 할 수 있다. 또한 상기 폴리펩티드는 폴리펩티드를 확인, 정제, 또는 합성할 수 있도록 다른 서열 또는 링커와 컨쥬게이트 될 수 있다.In addition, variants may contain deletions or additions of amino acids that have minimal effect on the properties and secondary structure of the polypeptide. For example, the polypeptide may be conjugated with a signal (or leader) sequence at the N-terminus of the protein involved in the transfer of the protein either co-translationally or post-translationally. The polypeptide may also be conjugated with other sequences or linkers to enable identification, purification, or synthesis of the polypeptide.
일 구현예로, 상기 변이체는 서열번호 1의 아미노산 서열의 N-말단으로부터 653번째 위치에 상응하는 아미노산이 메티오닌으로 치환된, 글루타메이트-시스테인 리가아제(glutamate-cysteine ligase) 변이체 일 수 있다. 일 구현예로, 상기 변이체는 서열번호 1의 아미노산 서열에서 653번 위치에 상응하는 글리신이 메티오닌으로 치환된 변이체 일 수 있으나 이에 제한되지 않는다.In one embodiment, the variant may be a glutamate-cysteine ligase (glutamate-cysteine ligase) variant in which the amino acid corresponding to the 653th position from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is substituted with methionine. In one embodiment, the variant may be a variant in which glycine corresponding to position 653 in the amino acid sequence of SEQ ID NO: 1 is substituted with methionine, but is not limited thereto.
본 출원에서, '다른 아미노산으로 치환'은 치환 전의 아미노산과 다른 아미노산이면 제한되지 않는다. 한편, 본 출원에서 '특정 아미노산이 치환되었다'고 표현하는 경우, 다른 아미노산으로 치환되었다고 별도로 표기하지 않더라도 치환 전의 아미노산과 다른 아미노산으로 치환되는 것임은 자명하다.본 출원에서 용어 “상응하는 위치(corresponding position)”는, 단백질 또는 폴리펩티드에서 열거되는 위치의 아미노산 잔기이거나, 또는 단백질 또는 폴리펩티드에서 열거되는 잔기와 유사하거나 동일하거나 상동한 아미노산 잔기를 지칭한다. 본 출원에 사용된 "상응 영역"은 일반적으로 관련 단백질 또는 레퍼런스 단백질에서의 유사하거나 대응되는 위치를 지칭한다.In the present application, 'substitution with another amino acid' is not limited as long as it is an amino acid different from the amino acid before the substitution. On the other hand, in the present application, when it is expressed that 'a specific amino acid has been substituted', it is obvious that the amino acid is substituted with an amino acid different from the amino acid before the substitution, even if it is not separately indicated that it is substituted with another amino acid. In the present application, the term "corresponding position (corresponding position)" refers to an amino acid residue at a listed position in a protein or polypeptide, or an amino acid residue similar to, identical to, or homologous to, a listed residue in a protein or polypeptide. As used herein, "corresponding region" generally refers to a similar or corresponding position in a related protein or reference protein.
본 출원에서, 본 출원에 사용되는 단백질 내의 아미노산 잔기 위치에 특정 넘버링이 사용될 수 있다. 예를 들면, 비교하고자 하는 대상 단백질과 본 출원의 단백질의 폴리펩티드 서열을 정렬함으로써, 본 출원의 단백질의 아미노산 잔기 위치에 상응하는 위치에 대해 재넘버링 하는 것이 가능하다.In the present application, specific numbering may be used for amino acid residue positions in proteins used in this application. For example, by aligning the polypeptide sequence of the protein of the present application with the target protein to be compared, it is possible to renumber the position corresponding to the amino acid residue position of the protein of the present application.
본 출원의 서열번호 1의 아미노산 서열의 N-말단으로부터 653번째 위치에 상응하는 아미노산이 메티오닌으로 치환된, 글루타메이트-시스테인 리가아제 변이체는, 서열번호 1의 아미노산 서열 또는 이와 80%, 85%, 90%, 95%, 96%, 97%, 98%, 또는 99% 이상의 상동성 또는 동일성을 갖는 아미노산 서열 중, 서열번호 1의 653번째 위치에 상응하는 아미노산이 메티오닌으로 치환된 단백질일 수 있다. 이와 같은 변이체는 서열번호 1과 80%, 85%, 90%, 95%, 96%, 97%, 98%, 또는 99% 이상의 상동성 또는 동일성을 가지고, 서열번호 1과 100% 미만의 상동성 또는 동일성을 가지는 변이체일 수 있으나 이에 제한되지 않는다. The amino acid corresponding to position 653 from the N-terminus of the amino acid sequence of SEQ ID NO: 1 of the present application is substituted with methionine, the glutamate-cysteine ligase variant is the amino acid sequence of SEQ ID NO: 1 or 80%, 85%, 90% thereof %, 95%, 96%, 97%, 98%, or a protein in which the amino acid corresponding to position 653 of SEQ ID NO: 1 is substituted with methionine among amino acid sequences having homology or identity of at least 99%. Such a variant has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity with SEQ ID NO: 1, and less than 100% homology with SEQ ID NO: 1 Or it may be a variant having the same identity, but is not limited thereto.
본 출원의 서열번호 1의 아미노산 서열의 N-말단으로부터 653번째 위치에 상응하는 아미노산이 메티오닌으로 치환된, 글루타메이트-시스테인 리가아제 변이체는, 서열번호 3의 아미노산 서열을 포함하는 것일 수 있다. 구체적으로 서열번호 3의 아미노산 서열로 필수적으로 구성되는 (consisting essentially of) 것일 수 있고, 보다 구체적으로는 서열번호 3 중 어느 하나의 아미노산 서열로 구성되는 것일 수 있으나 이에 제한되지 않는다.The amino acid corresponding to the 653th position from the N-terminus of the amino acid sequence of SEQ ID NO: 1 of the present application is substituted with methionine, the glutamate-cysteine ligase variant may include the amino acid sequence of SEQ ID NO: 3. Specifically, it may consist essentially of the amino acid sequence of SEQ ID NO: 3, and more specifically, it may consist of the amino acid sequence of any one of SEQ ID NO: 3, but is not limited thereto.
또한 상기 변이체는 서열번호 3의 아미노산 서열을 포함하거나, 또는 상기 아미노산 서열에서 653번 아미노산은 고정되고(즉, 변이체의 아미노산 서열에서 상기 서열번호 3의 653번 위치에 상응하는 아미노산은 상기 서열번호 3의 653번 위치의 아미노산과 동일하고), 이와 80% 이상의 상동성 또는 동일성을 갖는 아미노산 서열을 포함할 수 있으나, 이에 제한되는 것은 아니다. In addition, the variant includes the amino acid sequence of SEQ ID NO: 3, or amino acid 653 in the amino acid sequence is fixed (ie, the amino acid corresponding to position 653 of SEQ ID NO: 3 in the amino acid sequence of SEQ ID NO: 3 It is the same as the amino acid at position 653 of), and may include an amino acid sequence having 80% or more homology or identity therewith, but is not limited thereto.
구체적으로 본 출원의 상기 변이체는 서열번호 3 및 상기 서열번호 3 의 아미노산 서열과 적어도 80%, 90%, 95%, 96%, 97%, 98%, 또는 99% 상동성 또는 동일성을 가지는 폴리펩티드를 포함할 수 있다. 또한, 이러한 상동성 또는 동일성을 가지며 상기 변이체에 상응하는 효능을 나타내는 아미노산 서열이라면, 653번 위치 이외에, 일부 서열이 결실, 변형, 치환 또는 부가된 아미노산 서열을 갖는 단백질도 본 출원의 범위에 포함됨은 자명하다. Specifically, the variant of the present application is a polypeptide having at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% homology or identity to SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 3 may include In addition, if it is an amino acid sequence having such homology or identity and exhibiting efficacy corresponding to the variant, a protein having an amino acid sequence in which some sequence is deleted, modified, substituted or added other than position 653 is also included in the scope of the present application. self-evident
본 출원에서 용어, ‘상동성 (homology)’ 또는 ‘동일성 (identity)’은 두 개의 주어진 아미노산 서열 또는 염기 서열과 관련된 정도를 의미하며 백분율로 표시될 수 있다. 용어 상동성 및 동일성은 종종 상호교환적으로 이용될 수 있다.As used herein, the term 'homology' or 'identity' refers to a degree related to two given amino acid sequences or nucleotide sequences and may be expressed as a percentage. The terms homology and identity can often be used interchangeably.
보존된(conserved) 폴리뉴클레오티드 또는 폴리펩티드의 서열 상동성 또는 동일성은 표준 배열 알고리즘에 의해 결정되며, 사용되는 프로그램에 의해 확립된 디폴트 갭 페널티가 함께 이용될 수 있다. 실질적으로, 상동성을 갖거나(homologous) 또는 동일한(identical) 서열은 일반적으로 서열 전체 또는 전체-길이의 적어도 약 50%, 60%, 70%, 80% 또는 90%를 따라 중간 또는 높은 엄격한 조건(stringent conditions)에서 하이브리드할 수 있다. 하이브리드화는 폴리뉴클레오티드에서 일반 코돈 또는 코돈 축퇴성을 고려한 코돈을 함유하는 폴리뉴클레오티드 역시 포함됨이 자명하다.Sequence homology or identity of a conserved polynucleotide or polypeptide is determined by standard alignment algorithms, with default gap penalties established by the program used may be used. Substantially, homologous or identical sequences generally have moderate or high stringency conditions along at least about 50%, 60%, 70%, 80% or 90% of the entire or full-length sequence. It can hybridize under stringent conditions. It is obvious that hybridization also includes polynucleotides containing common codons in polynucleotides or codons taking codon degeneracy into account.
임의의 두 폴리뉴클레오티드 또는 폴리펩티드 서열이 상동성, 유사성 또는 동일성을 갖는지 여부는, 예를 들어, Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444에서와 같은 디폴트 파라미터를 이용하여 "FASTA" 프로그램과 같은 공지의 컴퓨터 알고리즘을 이용하여 결정될 수 있다. 또는, EMBOSS 패키지의 니들만 프로그램(EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277)(버전 5.0.0 또는 이후 버전)에서 수행되는 바와 같은, 니들만-운치(Needleman-Wunsch) 알고리즘(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453)이 사용되어 결정될 수 있다(GCG 프로그램 패키지 (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego,1994, 및 [CARILLO et al.](1988) SIAM J Applied Math 48: 1073을 포함한다). 예를 들어, 국립 생물공학 정보 데이터베이스 센터의 BLAST, 또는 ClustalW를 이용하여 상동성, 유사성 또는 동일성을 결정할 수 있다.Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined, for example, by Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444, using a known computer algorithm such as the “FASTA” program. or, as performed in the Needleman program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (version 5.0.0 or later), Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) can be used to determine (GCG program package (Devereux, J., et al, Nucleic Acids) Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, [S.] [F.,] [ET AL, J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, Martin J. Bishop , [ED.,] Academic Press, San Diego, 1994, and [CARILLO et al.] (1988) SIAM J Applied Math 48: 1073).For example, BLAST of the National Center for Biotechnology Information Database, or ClustalW can be used to determine homology, similarity or identity.
폴리뉴클레오티드 또는 폴리펩티드의 상동성, 유사성 또는 동일성은, 예를 들어, Smith and Waterman, Adv. Appl. Math (1981) 2:482 에 공지된 대로, 예를 들면, Needleman et al. (1970), J Mol Biol. 48:443과 같은 GAP 컴퓨터 프로그램을 이용하여 서열 정보를 비교함으로써 결정될 수 있다. 요약하면, GAP 프로그램은 두 서열 중 더 짧은 것에서의 기호의 전체 수로, 유사한 배열된 기호(즉, 뉴클레오티드 또는 아미노산)의 수를 나눈 값으로 정의할 수 있다. GAP 프로그램을 위한 디폴트 파라미터는 (1) 이진법 비교 매트릭스(동일성을 위해 1 그리고 비-동일성을 위해 0의 값을 함유함) 및 Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979)에 의해 개시된 대로, Gribskov et al(1986) Nucl. Acids Res. 14: 6745의 가중된 비교 매트릭스 (또는 EDNAFULL (NCBI NUC4.4의 EMBOSS 버전) 치환 매트릭스); (2) 각 갭을 위한 3.0의 페널티 및 각 갭에서 각 기호를 위한 추가의 0.10 페널티 (또는 갭 개방 패널티 10, 갭 연장 패널티 0.5); 및 (3) 말단 갭을 위한 무 페널티를 포함할 수 있다.Homology, similarity or identity of polynucleotides or polypeptides is described, for example, in Smith and Waterman, Adv. Appl. Math (1981) 2:482, see, for example, Needleman et al. (1970), J Mol Biol. can be determined by comparing sequence information using a GAP computer program such as 48:443. In summary, a GAP program can be defined as the total number of symbols in the shorter of two sequences divided by the number of similarly aligned symbols (ie, nucleotides or amino acids). Default parameters for the GAP program are: (1) a binary comparison matrix (containing values of 1 for identity and 0 for non-identity) and Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation , pp. 353-358 (1979), Gribskov et al (1986) Nucl. Acids Res. 14: weighted comparison matrix of 6745 (or EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix); (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap (or a gap opening penalty of 10, a gap extension penalty of 0.5); and (3) no penalty for end gaps.
또한, 임의의 두 폴리뉴클레오티드 또는 폴리펩티드 서열이 상동성, 유사성 또는 동일성을 갖는지 여부는 정의된 엄격한 조건하에서 써던 혼성화 실험에 의해 서열을 비교함으로써 확인할 수 있으며, 정의되는 적절한 혼성화 조건은 해당 기술 범위 내이고, 당업자에게 잘 알려진 방법(예컨대, J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York)으로 결정될 수 있다.In addition, whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be confirmed by comparing the sequences by Southern hybridization experiments under defined stringent conditions, and the defined appropriate hybridization conditions are within the scope of the art and , methods well known to those of skill in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; F.M. Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York).
본 출원의 서열번호 1의 아미노산 서열의 N-말단으로부터 653번째 위치에 상응하는 아미노산이 메티오닌으로 치환된 글루타메이트-시스테인 리가아제 변이체는 추가적으로, 서열번호 1의 아미노산 서열의 N-말단으로부터 86번 위치에 상응하는 아미노산이 다른 아미노산으로 치환된 변이를 포함할 수 있다. Glutamate-cysteine ligase variant in which the amino acid corresponding to position 653 from the N-terminus of the amino acid sequence of SEQ ID NO: 1 of the present application is substituted with methionine is additionally at position 86 from the N-terminus of the amino acid sequence of SEQ ID NO: 1 It may contain mutations in which the corresponding amino acid is substituted for another amino acid.
구체적으로, 상기 변이체는 86번 위치에 상응하는 시스테인이 다른 아미노산으로 치환된 변이를 포함할 수 있고, 일 예로 아르기닌으로 치환된 변이를 포함하는 것일 수 있다. Specifically, the variant may include a mutation in which the cysteine corresponding to position 86 is substituted with another amino acid, and may include, for example, a mutation in which arginine is substituted.
일 예로, 상기 변이체는 서열번호 13의 아미노산 서열을 포함하거나, 이로 필수적으로 구성되거나 혹은 이로 구성되는 것일 수 있다. 그러나 이에 제한되지 않는다.For example, the variant may include, consist essentially of, or consist of the amino acid sequence of SEQ ID NO: 13. However, it is not limited thereto.
본 출원의 다른 하나의 양태는 상기 변이체를 코딩하는 폴리뉴클레오티드를 제공할 수 있다. Another aspect of the present application may provide a polynucleotide encoding the variant.
본 출원에서 용어, "폴리뉴클레오티드"는 뉴클레오티드 단위체(monomer)가 공유결합에 의해 길게 사슬모양으로 이어진 뉴클레오티드의 중합체(polymer)로 일정한 길이 이상의 DNA 또는 RNA 가닥을 의미한다. As used herein, the term "polynucleotide" refers to a DNA or RNA strand of a certain length or more as a polymer of nucleotides in which nucleotide monomers are connected in a long chain form by covalent bonds.
본 출원의 글루타메이트-시스테인 리가아제를 코딩하는 유전자는 gsh1 유전자일 수 있다. The gene encoding the glutamate-cysteine ligase of the present application may be a gsh1 gene.
상기 유전자는 효모 유래 일 수 있다. 구체적으로는 사카로마이세스(Saccharomyces) 속, 보다 구체적으로는 사카로마이세스 세레비지애(Saccharomyces cerevisiae) 유래일 수 있다. 구체적으로는 사카로마이세스 세레비지애 유래의 글루타메이트-시스테인 리가아제 활성을 갖는 폴리펩티드를 코딩하는 것이면 제한없이 포함하고, 일 구현예로 서열번호 1의 아미노산 서열을 코딩하는 유전자일 수 있으며, 일 구현예로 서열번호 2의 염기서열을 포함하는 것일 수 있으나 이에 제한되지 않는다. The gene may be derived from yeast. Specifically, it may be derived from Saccharomyces genus, more specifically Saccharomyces cerevisiae. Specifically, glutamate derived from Saccharomyces cerevisiae includes without limitation as long as it encodes a polypeptide having cysteine ligase activity, and in one embodiment may be a gene encoding the amino acid sequence of SEQ ID NO: 1, one embodiment For example, it may include the nucleotide sequence of SEQ ID NO: 2, but is not limited thereto.
본 출원의 단백질 변이체를 코딩하는 폴리뉴클레오티드는, 본 출원의 글루타메이트-시스테인 리가아제 변이체 및 이에 상응하는 활성을 갖는 폴리펩티드를 코딩하는 폴리뉴클레오티드라면 제한없이 포함될 수 있다. The polynucleotide encoding the protein variant of the present application may be included without limitation as long as it is a polynucleotide encoding a glutamate-cysteine ligase variant of the present application and a polypeptide having a corresponding activity.
본 출원의 글루타메이트-시스테인 리가아제 및 이의 변이체를 코딩하는 폴리뉴클레오티드는 코돈의 축퇴성(degeneracy)으로 인하여 또는 상기 폴리펩티드를 발현시키고자 하는 생물에서 선호되는 코돈을 고려하여, 폴리펩티드의 아미노산 서열을 변화시키지 않는 범위 내에서 코딩 영역에 다양한 변형이 이루어질 수 있다. The polynucleotide encoding the glutamate-cysteine ligase and variants thereof of the present application does not change the amino acid sequence of the polypeptide due to codon degeneracy or in consideration of codons preferred in the organism in which the polypeptide is to be expressed. Various modifications may be made to the coding region within the non-limiting range.
구체적으로, 본 출원의 단백질 변이체를 코딩하는 폴리뉴클레오티드는, 서열번호 1의 아미노산 서열에서 653번째 위치에 상응하는 아미노산이 메티오닌으로 치환된 단백질 변이체를 코딩하는 폴리뉴클레오티드 서열이라면 제한 없이 포함할 수 있다. 예를 들어, 본 출원의 단백질 변이체를 코딩하는 폴리뉴클레오티드는 본 출원의 단백질 변이체, 구체적으로는 상기 서열번호 3의 아미노산 서열을 포함하는 단백질 또는 이와 상동성 또는 동일성을 갖는 폴리펩티드를 코딩하는 폴리뉴클레오티드 서열일 수 있으나, 이에 제한되지 않는다. 상기 상동성 또는 동일성에 대해서는 전술한 바와 같다. Specifically, the polynucleotide encoding the protein variant of the present application may include without limitation as long as it is a polynucleotide sequence encoding a protein variant in which the amino acid corresponding to position 653 in the amino acid sequence of SEQ ID NO: 1 is substituted with methionine. For example, the polynucleotide encoding the protein variant of the present application is a polynucleotide sequence encoding the protein variant of the present application, specifically, a protein comprising the amino acid sequence of SEQ ID NO: 3 or a polypeptide having homology or identity thereto may be, but is not limited thereto. The homology or identity is the same as described above.
또한, 본 출원의 단백질 변이체를 코딩하는 폴리뉴클레오티드는 공지의 유전자 서열로부터 제조될 수 있는 프로브, 예를 들면, 상기 염기 서열의 전체 또는 일부에 대한 상보 서열과 엄격한 조건 하에 하이브리드화하여, 서열번호 1의 아미노산 서열에서 653번째 위치에 상응하는 아미노산이 메티오닌으로 치환된 단백질 변이체를 코딩하는 서열이라면 제한 없이 포함할 수 있다.In addition, the polynucleotide encoding the protein variant of the present application is hybridized under stringent conditions with a probe that can be prepared from a known gene sequence, for example, a sequence complementary to all or part of the base sequence, SEQ ID NO: 1 Any sequence encoding a protein variant in which the amino acid corresponding to position 653 in the amino acid sequence of is substituted with methionine may be included without limitation.
상기 “엄격한 조건(stringent condition)”이란 폴리뉴클레오티드 간의 특이적 혼성화를 가능하게 하는 조건을 의미한다. 이러한 조건은 문헌(예컨대, J. Sambrook et al., 1989, 상동)에 구체적으로 기재되어 있다. 예를 들어, 상동성 또는 동일성이 높은 폴리뉴클레오티드끼리, 40% 이상, 구체적으로 90% 이상, 보다 구체적으로 95% 이상, 96% 이상, 97% 이상, 98% 이상, 더욱 구체적으로 99% 이상의 상동성 또는 동일성을 갖는 폴리뉴클레오티드끼리 하이브리드화하고, 그보다 상동성 또는 동일성이 낮은 폴리뉴클레오티드끼리 하이브리드화하지 않는 조건, 또는 통상의 써던 하이브리드화(southern hybridization)의 세척 조건인 60℃, 1×SSC, 0.1% SDS, 구체적으로 60℃, 0.1×SSC, 0.1% SDS, 보다 구체적으로 68℃, 0.1×SSC, 0.1% SDS에 상당하는 염 농도 및 온도에서, 1회, 구체적으로 2회 내지 3회 세정하는 조건을 열거할 수 있다.The “stringent condition” refers to a condition that enables specific hybridization between polynucleotides. These conditions are specifically described in the literature (eg, J. Sambrook et al., 1989, supra). For example, polynucleotides with high homology or identity are 40% or more, specifically 90% or more, more specifically 95% or more, 96% or more, 97% or more, 98% or more, and more specifically 99% or more. Conditions in which polynucleotides having homology or identity hybridize with each other and polynucleotides with lower homology or identity do not hybridize, or wash conditions of conventional Southern hybridization at 60° C., 1×SSC, 0.1 at a salt concentration and temperature equivalent to % SDS, specifically 60° C., 0.1×SSC, 0.1% SDS, more specifically 68° C., 0.1×SSC, 0.1% SDS, washing once, specifically 2 to 3 times Conditions can be enumerated.
혼성화는 비록 혼성화의 엄격도에 따라 염기 간의 미스매치(mismatch)가 가능할지라도, 두 개의 핵산이 상보적 서열을 가질 것을 요구한다. 용어, “상보적”은 서로 혼성화가 가능한 뉴클레오티드 염기 간의 관계를 기술하는데 사용된다. 예를 들면, DNA에 관하여, 아데노신은 티민에 상보적이며 시토신은 구아닌에 상보적이다. 따라서, 본 출원의 폴리뉴클레오티드는 또한 실질적으로 유사한 핵산 서열뿐만 아니라 전체 서열에 상보적인 단리된 핵산 단편을 포함할 수 있다.Hybridization requires that two nucleic acids have complementary sequences, although mismatch between bases is possible depending on the stringency of hybridization. The term “complementary” is used to describe the relationship between nucleotide bases capable of hybridizing to each other. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine. Accordingly, the polynucleotides of the present application may also include substantially similar nucleic acid sequences as well as isolated nucleic acid fragments complementary to the overall sequence.
구체적으로, 상동성 또는 동일성을 가지는 폴리뉴클레오티드는 55 ℃의 Tm 값에서 혼성화 단계를 포함하는 혼성화 조건을 사용하고 상술한 조건을 사용하여 탐지할 수 있다. 또한, 상기 Tm 값은 60 ℃, 63 ℃ 또는 65 ℃일 수 있으나, 이에 제한되는 것은 아니고 그 목적에 따라 당업자에 의해 적절히 조절될 수 있다.Specifically, polynucleotides having homology or identity can be detected using hybridization conditions including a hybridization step at a Tm value of 55° C. and using the above-described conditions. In addition, the Tm value may be 60 °C, 63 °C, or 65 °C, but is not limited thereto and may be appropriately adjusted by those skilled in the art according to the purpose.
폴리뉴클레오티드를 혼성화하는 적절한 엄격도는 폴리뉴클레오티드의 길이 및 상보성 정도에 의존하고 변수는 해당기술분야에 잘 알려져 있다(Sambrook et al., 1989, supra, 9.50-9.51, 11.7-11.8 참조).The appropriate stringency for hybridizing polynucleotides depends on the length of the polynucleotide and the degree of complementarity, and the parameters are well known in the art (see Sambrook et al., 1989, supra, 9.50-9.51, 11.7-11.8).
본 출원의 다른 하나의 양태는 상기 단백질 변이체를 코딩하는 폴리뉴클레오티드를 포함하는 벡터를 제공할 수 있다.Another aspect of the present application may provide a vector comprising a polynucleotide encoding the protein variant.
본 출원에서 사용된 용어 "벡터"는 적합한 숙주 내에서 목적 폴리펩티드를 발현시킬 수 있도록 적합한 발현조절영역(또는 발현조절서열)에 작동 가능하게 연결된 상기 목적 폴리펩티드를 코딩하는 폴리뉴클레오티드의 염기서열을 함유하는 DNA 제조물을 의미한다. 상기 발현조절영역은 전사를 개시할 수 있는 프로모터, 그러한 전사를 조절하기 위한 임의의 오퍼레이터 서열, 적합한 mRNA 리보좀 결합부위를 코딩하는 서열, 및 전사 및 해독의 종결을 조절하는 서열을 포함할 수 있다. 벡터는 적당한 숙주세포 내로 형질전환된 후, 숙주 게놈과 무관하게 복제되거나 기능할 수 있으며, 게놈 그 자체에 통합될 수 있다.The term "vector" as used in the present application refers to a polynucleotide encoding a target polypeptide operably linked to a suitable expression control region (or expression control sequence) so that the target polypeptide can be expressed in a suitable host. DNA preparations. The expression control region may include a promoter capable of initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. After transformation into an appropriate host cell, the vector can replicate or function independently of the host genome, and can be integrated into the genome itself.
일례로 세포 내 염색체 삽입용 벡터를 통해 염색체 내에 목적 단백질을 코딩하는 폴리뉴클레오티드를 변이된 폴리뉴클레오티드로 교체시킬 수 있다. 상기 폴리뉴클레오티드의 염색체 내로의 삽입은 당업계에 알려진 임의의 방법, 예를 들면, 상동재조합에 의하여 이루어질 수 있으나, 이에 한정되지는 않는다. 상기 염색체 삽입 여부를 확인하기 위한 선별 마커(selection marker)를 추가로 포함할 수 있다. 선별 마커는 벡터로 형질전환된 세포를 선별, 즉 목적 핵산 분자의 삽입 여부를 확인하기 위한 것으로, 약물 내성, 영양 요구성, 세포 독성제에 대한 내성 또는 표면 단백질의 발현과 같은 선택가능 표현형을 부여하는 마커들이 사용될 수 있다. 선택제(selective agent)가 처리된 환경에서는 선별 마커를 발현하는 세포만 생존하거나 다른 표현 형질을 나타내므로, 형질전환된 세포를 선별할 수 있다.For example, a polynucleotide encoding a target protein in a chromosome may be replaced with a mutated polynucleotide through a vector for intracellular chromosome insertion. Insertion of the polynucleotide into a chromosome may be performed by any method known in the art, for example, homologous recombination, but is not limited thereto. It may further include a selection marker (selection marker) for confirming whether the chromosome is inserted. The selection marker is used to select cells transformed with the vector, that is, to confirm whether a target nucleic acid molecule is inserted, and to confer a selectable phenotype such as drug resistance, auxotrophicity, resistance to cytotoxic agents, or surface protein expression. markers may be used. In an environment treated with a selective agent, only the cells expressing the selectable marker survive or exhibit other expression traits, so that the transformed cells can be selected.
본 출원에서 사용되는 벡터는 특별히 한정되지 않으며, 당업계에 알려진 임의의 벡터를 이용할 수 있다. 효모 발현 벡터는 조합 효모 플라스미드(YIp: integrative yeast plasmid) 및 염색체 외 플라스미드 벡터(extrachromosomal plasmid vector)가 모두 가능하다. 상기 염색체 외 플라스미드 벡터는 에피솜 효모 플라스미드(YEp: episomal yeast plasmid), 복제 효모 플라스미드(YRp: replicative yeast plasmid) 및 효모 중심체 플라스미드(YCp: yeast centromer plasmid)를 포함할 수 있다. 또한, 인위적 효모 염색체들(YACs: artificial yeast chromosomes)도 본 출원의 벡터로 이용이 가능하다. 구체적인 예로서, 이용 가능한 벡터는 pESCHIS, pESC-LEU, pESC-TRP, pESC-URA, Gateway pYES-DEST52, pAO815, pGAPZ A, pGAPZ B, pGAPZ C, pGAPα A, pGAPα B, pGAPα C, pPIC3.5K, pPIC6 A, pPIC6 B, pPIC6 C, pPIC6α A, pPIC6α B, pPIC6α C, pPIC9K, pYC2/CT, pYD1 Yeast Display Vector, pYES2, pYES2/CT, pYES2/NT A, pYES2/NT B, pYES2/NT C, pYES2/CT, pYES2.1, pYES-DEST52, pTEF1/Zeo, pFLD1, PichiaPinkTM, p427-TEF, p417-CYC, pGAL-MF, p427-TEF, p417-CYC, PTEF-MF, pBY011, pSGP47, pSGP46, pSGP36, pSGP40, ZM552, pAG303GAL-ccdB, pAG414GAL-ccdB, pAS404, pBridge, pGAD-GH, pGAD T7, pGBK T7, pHIS-2, pOBD2, pRS408, pRS410, pRS418, pRS420, pRS428, yeast micron A form, pRS403, pRS404, pRS405, pRS406, pYJ403, pYJ404, pYJ405 및 pYJ406를 포함하나, 이에 한정되는 것은 아니다.The vector used in the present application is not particularly limited, and any vector known in the art may be used. The yeast expression vector can be both an integrative yeast plasmid (YIp) and an extrachromosomal plasmid vector. The extrachromosomal plasmid vector may include an episomal yeast plasmid (YEp), a replicative yeast plasmid (YRp), and a yeast centromer plasmid (YCp). In addition, artificial yeast chromosomes (YACs) can also be used as the vector of the present application. As a specific example, available vectors are pESCHIS, pESC-LEU, pESC-TRP, pESC-URA, Gateway pYES-DEST52, pAO815, pGAPZ A, pGAPZ B, pGAPZ C, pGAPa A, pGAPa B, pGAPa C, pPIC3.5K , pPIC6 A, pPIC6 B, pPIC6 C, pPIC6α A, pPIC6α B, pPIC6α C, pPIC9K, pYC2/CT, pYD1 Yeast Display Vector, pYES2, pYES2/CT, pYES2/NT A, pYES2/NT B, pYES2/NT C , pYES2/CT, pYES2.1, pYES-DEST52, pTEF1/Zeo, pFLD1, PichiaPinkTM, p427-TEF, p417-CYC, pGAL-MF, p427-TEF, p417-CYC, PTEF-MF, pBY011, pSGP47, pSGP46 , pSGP36, pSGP40, ZM552, pAG303GAL-ccdB, pAG414GAL-ccdB, pAS404, pBridge, pGAD-GH, pGAD T7, pGBK T7, pHIS-2, pOBD2, pRS408, pRS410, pRS418, pRS420, pRS428, yeast micron A form pRS403, pRS404, pRS405, pRS406, pYJ403, pYJ404, pYJ405 and pYJ406.
본 출원에서 용어 "형질전환"은 표적 단백질을 코딩하는 폴리뉴클레오티드를 포함하는 벡터를 숙주세포 혹은 미생물 내에 도입하여 숙주세포 내에서 상기 폴리뉴클레오티드가 코딩하는 단백질이 발현할 수 있도록 하는 것을 의미한다. 형질전환된 폴리뉴클레오티드는 숙주세포 내에서 발현될 수 있기만 한다면, 숙주세포의 염색체 내에 삽입되어 위치하거나 염색체 외에 위치하거나 상관없이 이들 모두를 포함할 수 있다. 또한, 상기 폴리뉴클레오티드는 목적 단백질을 코딩하는 DNA 및 RNA를 포함한다. 상기 폴리뉴클레오티드는 숙주세포 내로 도입되어 발현될 수 있는 것이면, 어떠한 형태로도 도입될 수 있다. 예를 들면, 상기 폴리뉴클레오티드는 자체적으로 발현되는데 필요한 모든 요소를 포함하는 유전자 구조체인 발현 카세트(expression cassette)의 형태로 숙주세포에 도입될 수 있다. 상기 발현 카세트는 통상 상기 폴리뉴클레오티드에 작동 가능하게 연결되어 있는 프로모터(promoter), 전사 종결신호, 리보좀 결합부위 및 번역 종결신호를 포함할 수 있다. 상기 발현 카세트는 자체 복제가 가능한 발현 벡터 형태일 수 있다. 또한, 상기 폴리뉴클레오티드는 그 자체의 형태로 숙주세포에 도입되어 숙주세포에서 발현에 필요한 서열과 작동 가능하게 연결되어 있는 것일 수도 있으며, 이에 제한되지 않는다.In the present application, the term “transformation” refers to introducing a vector including a polynucleotide encoding a target protein into a host cell or microorganism so that the protein encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotide may include all of them regardless of whether they are inserted into the chromosome of the host cell or located extrachromosomally, as long as they can be expressed in the host cell. In addition, the polynucleotide includes DNA and RNA encoding a target protein. The polynucleotide may be introduced in any form as long as it can be introduced and expressed into a host cell. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct including all elements necessary for self-expression. The expression cassette may include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replication. In addition, the polynucleotide may be introduced into a host cell in its own form and operably linked to a sequence required for expression in the host cell, but is not limited thereto.
또한, 상기에서 용어 "작동 가능하게 연결"된 것이란 본 출원의 목적 폴리펩티드를 코딩하는 폴리뉴클레오티드의 전사를 개시 및 매개하도록 하는 프로모터 서열과 상기 유전자 서열이 기능적으로 연결되어 있는 것을 의미한다.In addition, the term “operably linked” as used herein means that a promoter sequence that initiates and mediates transcription of a polynucleotide encoding a target polypeptide of the present application and the gene sequence are functionally linked.
본 출원의 벡터를 형질전환 시키는 방법은 핵산을 세포 내로 도입하는 어떤 방법도 포함되며, 숙주세포에 따라 당 분야에서 공지된 바와 같이 적합한 표준 기술을 선택하여 수행할 수 있다. 예를 들어, 전기천공법(electroporation), 인산칼슘(CaPO4) 침전, 염화칼슘(CaCl2) 침전, 미세주입법(microinjection), 폴리에틸렌 글리콜(PEG)법, DEAE-덱스트란법, 양이온 리포좀법, 및 초산 리튬-DMSO법 등이 있으나, 이에 제한되지 않는다.The method for transforming the vector of the present application includes any method of introducing a nucleic acid into a cell, and may be performed by selecting a suitable standard technique as known in the art depending on the host cell. For example, electroporation, calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and Lithium acetate-DMSO method and the like, but is not limited thereto.
본 출원은 상기 변이체; 상기 변이체를 코딩하는 폴리뉴클레오티드; 및 상기 폴리뉴클레오티드를 포함하는 벡터 중 어느 하나 이상을 포함하여, 글루타치온을 생산하는 미생물을 제공할 수 있다.The present application relates to the variant; a polynucleotide encoding the variant; And it may include any one or more of vectors including the polynucleotide, to provide a microorganism producing glutathione.
본 출원에서 용어 "미생물"은 야생형 미생물이나, 자연적 또는 인위적으로 유전적 변형이 일어난 미생물을 모두 포함하며, 외부 유전자가 삽입되거나 내재적 유전자의 활성이 강화되거나 약화되는 등의 원인으로 인해서 특정 기작이 약화되거나 강화된 미생물을 모두 포함하는 개념이다. 본 출원에서 미생물은 본 출원의 글루타메이트-시스테인 리가아제 변이체가 도입되거나 포함되는 미생물이라면 제한 없이 포함될 수 있다.In the present application, the term "microorganism" includes both wild-type microorganisms and microorganisms in which genetic modification has occurred, either naturally or artificially, and a specific mechanism is weakened due to causes such as insertion of an external gene or enhanced or weakened activity of an intrinsic gene. It is a concept that includes all microorganisms that have been or have been fortified. The microorganism in the present application may be included without limitation as long as it is a microorganism into which the glutamate-cysteine ligase variant of the present application is introduced or included.
상기 미생물은, 목적 단백질을 코딩하는 유전자 또는 이를 포함하는 벡터로 형질전환되어, 예를 들어 목적 단백질을 발현하는 세포 또는 미생물로서, 본 출원의 목적상 상기 숙주세포 또는 미생물은 상기 글루타메이트-시스테인 리가아제 변이체를 포함하여 글루타치온을 생산할 수 있는 미생물이라면 모두 가능하다.The microorganism is transformed with a gene encoding a target protein or a vector comprising the same, for example, as a cell or microorganism expressing the target protein, and for the purpose of the present application, the host cell or microorganism is the glutamate-cysteine ligase Any microorganism capable of producing glutathione, including mutants, is possible.
본 출원의 "글루타치온(glutathione)"은 "글루타티온", "GSH"와 상호 교환적으로 사용되며, 글루타메이트(glutamate), 시스테인(Cysteine), 글리신(glycine) 의 세가지 아미노산으로 구성된 트리펩타이드를 의미한다. 글루타치온은 제약, 건강기능식품, 맛소재, 식품, 사료 첨가제, 화장품 등의 원료로 이용될 수 있으나, 이에 제한되지 않는다.In the present application, "glutathione" is used interchangeably with "glutathione" and "GSH", and refers to a tripeptide composed of three amino acids: glutamate, cysteine, and glycine. Glutathione may be used as a raw material for pharmaceuticals, health functional foods, flavoring materials, food, feed additives, cosmetics, etc., but is not limited thereto.
본 출원에서 "글루타치온을 생산하는 미생물"은 자연적 또는 인위적으로 유전적 변형이 일어난 미생물을 모두 포함하며, 외부 유전자가 삽입되거나 내재적 유전자의 활성이 강화되거나 불활성화되는 등의 원인으로 인해서 특정 기작이 약화되거나 강화된 미생물로서, 목적하는 글루타치온 생산을 위한 유전적 변이가 일어나거나 활성을 강화시킨 미생물일 수 있다. 본 출원의 목적상 상기 글루타치온을 생산하는 미생물은, 글루타메이트-시스테인 리가아제를 포함하여, 야생형이나 비변형 미생물과 비교하여 목적하는 글루타치온을 과량으로 생산할 수 있는 미생물을 의미할 수 있다. 상기 "글루타치온을 생산하는 미생물" 은 "글루타치온 생산 미생물","글루타치온 생산능을 갖는 미생물, "글루타치온 생산 균주", "글루타치온 생산능을 갖는 균주" 등의 용어와 혼용되어 사용될 수 있다. In the present application, "glutathione-producing microorganism" includes all microorganisms in which genetic modification has occurred, either naturally or artificially, and a specific mechanism is weakened due to causes such as insertion of an external gene or intensification or inactivation of the activity of an intrinsic gene As the modified or enhanced microorganism, it may be a microorganism having a genetic mutation or enhanced activity for the desired glutathione production. For the purpose of the present application, the glutathione-producing microorganism may refer to a microorganism capable of producing a desired glutathione in excess compared to a wild-type or unmodified microorganism, including glutamate-cysteine ligase. The "glutathione-producing microorganism" may be used interchangeably with terms such as "glutathione-producing microorganism", "microorganism having glutathione-producing ability," "glutathione-producing strain", "strain having glutathione-producing ability", and the like.
상기 글루타치온 생산 미생물은 글루타치온 생산이 가능하다면 그 종류가 특별히 제한되지는 않으나, 사카로마이세스 속 (the genus Saccharomyces) 미생물일 수 있고, 구체적으로는 사카로마이세스 세레비지애(Saccharomyces cerevisiae) 일 수 있으나, 이에 제한되지 않는다. The glutathione-producing microorganism is not particularly limited in its type as long as glutathione production is possible, but may be a microorganism of the genus Saccharomyces , specifically Saccharomyces cerevisiae ). However, the present invention is not limited thereto.
상기 변이체를 포함하는 글루타치온 생산 미생물의 모균주는, 글루타치온 생산이 가능한 것이면 특별히 제한되지 않는다. 상기 미생물은 추가로 글루타치온 생산능 증가를 위한 생합성경로 강화, 피드백 저해 해제, 분해경로 혹은 생합성 경로를 약화시키는 유전자 불활성화 등의 변이를 포함하는 것일 수 있고, 이러한 변이는 자연적인 것을 배제하는 것은 아니다. 일 구현예로, 상기 미생물은 글루타메이트-시스테인 리가아제의 발현조절영역에 글루타치온 생산능이 증가되도록 하는 변이를 포함하는 것일 수 있다. 이는 GSH1 ORF 상단의 -250(C→T), -252(G→A), -398(A→T), -399(A→C), -407(T→C) 및 -409(T→C) 중에서 선택되는 어느 하나 이상의 변이일 수 있다. 그러나, 이에 제한되지 않는다.The parent strain of the glutathione-producing microorganism including the mutant is not particularly limited as long as it is capable of producing glutathione. The microorganism may further include mutations such as enhancement of biosynthetic pathways for increasing glutathione production capacity, release of feedback inhibition, gene inactivation that weakens degradation pathways or biosynthetic pathways, and such mutations do not exclude natural ones . In one embodiment, the microorganism may include a mutation in the expression control region of glutamate-cysteine ligase to increase glutathione production ability. These are -250 (C→T), -252 (G→A), -398 (A→T), -399 (A→C), -407 (T→C) and -409 (T→) on top of the GSH1 ORF. It may be any one or more mutations selected from C). However, it is not limited thereto.
본 출원의 변이체; 상기 변이체를 코딩하는 폴리뉴클레오티드; 및 상기 폴리뉴클레오티드를 포함하는 벡터 중 어느 하나 이상을 포함하는 미생물은, 서열번호 1의 아미노산 서열에서 653번째 위치에 상응하는 아미노산이 메티오닌으로 치환된 글루타메이트-시스테인 리가아제 변이체를 발현하는 미생물일 수 있으나 이에 제한되지 않는다. variants of the present application; a polynucleotide encoding the variant; And the microorganism comprising any one or more of the vector containing the polynucleotide, glutamate-cysteine ligase variant in which the amino acid corresponding to position 653 in the amino acid sequence of SEQ ID NO: 1 is substituted with methionine may be a microorganism It is not limited thereto.
상기 글루타메이트-시스테인 리가아제 및 이의 변이체에 대해서는 전술한 바와 같다.The glutamate-cysteine ligase and its variants are as described above.
본 출원에서 용어, 단백질이 "발현되도록/되는"은 목적 단백질이 미생물 내에 도입되거나, 미생물 내에서 발현되도록 변형된 상태를 의미한다. 상기 목적 단백질이 미생물 내 존재하는 단백질인 경우 내재적 또는 변형전에 비하여 그 활성이 강화된 상태를 의미한다.As used herein, the term "to be/are" a protein refers to a state in which a target protein is introduced into a microorganism or modified to be expressed in the microorganism. When the target protein is a protein present in a microorganism, it refers to a state in which the activity is enhanced compared to before intrinsic or modification.
본 출원의 단백질 변이체를 발현하는 미생물은, 본 출원의 단백질 변이체를 발현하도록 변형된 미생물일 수 있고, 따라서 본 출원의 다른 하나의 양태는 본 출원의 단백질 변이체를 발현하는 미생물의 제조 방법을 제공한다.The microorganism expressing the protein variant of the present application may be a microorganism modified to express the protein variant of the present application, and thus another aspect of the present application provides a method for producing a microorganism expressing the protein variant of the present application. .
본 출원에서 "단백질의 도입"은, 미생물이 본래 가지고 있지 않았던 특정 단백질의 활성을 나타나게 되는 것 또는 해당 단백질의 내재적 활성 또는 변형 전 활성에 비하여 향상된 활성을 나타나게 되는 것을 의미한다. 예를 들어, 특정 단백질이 도입되거나, 특정 단백질을 코딩하는 폴리뉴클레오티드가 미생물 내 염색체로 도입되거나, 특정 단백질을 코딩하는 폴리뉴클레오티드를 포함하는 벡터가 미생물 내로 도입되어 이의 활성이 나타나는 것일 수 있다.In the present application, "introduction of protein" refers to exhibiting the activity of a specific protein that the microorganism did not originally have, or exhibiting improved activity compared to the intrinsic activity or activity prior to modification of the corresponding protein. For example, a specific protein is introduced, a polynucleotide encoding a specific protein is introduced into a chromosome in a microorganism, or a vector including a polynucleotide encoding a specific protein is introduced into a microorganism to exhibit its activity.
본 출원에서 용어, 폴리펩티드 또는 단백질 활성의 “강화”는, 폴리펩티드 또는 단백질의 활성이 내재적 활성에 비하여 증가되는 것을 의미한다. 상기 강화는 상향조절(up-regulation), 과발현(overexpression), 증가(increase) 등의 용어와 혼용될 수 있다. 여기서 증가는 본래 가지고 있지 않았던 활성을 나타내게 되는 것, 또는 내재적 활성 또는 변형 전 활성에 비하여 향상된 활성을 나타내게 되는 것을 모두 포함할 수 있다. 상기 “내재적 활성”은 자연적 또는 인위적 요인에 의한 유전적 변이로 형질이 변화하는 경우, 형질 변화 전 모균주 또는 비변형 미생물이 본래 가지고 있던 특정 폴리펩티드 또는 단백질의 활성을 의미한다. 이는 “변형 전 활성”과 혼용되어 사용될 수 있다. 폴리펩티드 또는 단백질의 활성이 내재적 활성에 비하여 “강화” 또는 “증가”한다는 것은, 형질 변화 전 모균주 또는 비변형 미생물이 본래 가지고 있던 특정 폴리펩티드 또는 단백질의 활성에 비하여 향상된 것을 의미한다. As used herein, the term “enhancement” of polypeptide or protein activity means that the activity of the polypeptide or protein is increased compared to the intrinsic activity. The reinforcement may be used interchangeably with terms such as up-regulation, overexpression, and increase. Here, the increase may include both exhibiting an activity that it did not originally have, or exhibiting an improved activity compared to intrinsic activity or activity before modification. The “intrinsic activity” refers to the activity of a specific polypeptide or protein originally possessed by the parent strain or unmodified microorganism before the transformation when the trait is changed due to genetic mutation caused by natural or artificial factors. This may be used interchangeably with “activity before modification”. “Enhancement” or “increase” in the activity of a polypeptide or protein compared to its intrinsic activity means that it is improved compared to the activity of a specific polypeptide or protein originally possessed by the parent strain or unmodified microorganism before transformation.
상기 “활성 증가”는 외래의 폴리펩티드 또는 단백질을 도입하거나, 내재적인 폴리펩티드 또는 단백질의 활성 강화를 통해 달성할 수 있으나, 구체적으로는 내재적인 폴리펩티드 또는 단백질의 활성 강화를 통해 달성하는 것일 수 있다. 상기 폴리펩티드 또는 단백질의 활성의 강화 여부는 해당 폴리펩티드 또는 단백질의 활성 정도, 발현량 또는 해당 단백질로부터 배출되는 산물의 양의 증가로부터 확인할 수 있다.The “increase in activity” may be achieved by introducing an exogenous polypeptide or protein or enhancing the activity of an endogenous polypeptide or protein, but specifically, it may be achieved through enhancing the activity of an endogenous polypeptide or protein. Whether the activity of the polypeptide or protein is enhanced can be confirmed from an increase in the activity level, expression level, or amount of a product excreted from the corresponding polypeptide or protein.
상기 폴리펩티드 또는 단백질의 활성의 강화는 당해 분야에 잘 알려진 다양한 방법의 적용이 가능하며, 목적 폴리펩티드 또는 단백질의 활성을 변형전 미생물보다 강화시킬 수 있는 한, 제한되지 않을 수 있다. 상기 방법은 이로 제한되는 것은 아니나, 분자생물학의 일상적 방법인 당업계의 통상의 기술자에게 잘 알려진 유전자 공학 및/또는 단백질 공학을 이용한 것일 수 있다(Sitnicka et al. Functional Analysis of Genes. Advances in Cell Biology. 2010, Vol. 2. 1-16, Sambrook et al. Molecular Cloning 2012 등).The enhancement of the activity of the polypeptide or protein can be applied by various methods well known in the art, and may not be limited as long as it can enhance the activity of the target polypeptide or protein compared to the microorganism before modification. The method is not limited thereto, but may use genetic engineering and/or protein engineering well known to those skilled in the art, which are routine methods of molecular biology (Sitnicka et al . Functional Analysis of Genes. Advances in Cell Biology). 2010, Vol. 2. 1-16, Sambrook et al . Molecular Cloning 2012 et al.).
상기 유전자 공학을 이용하여 폴리펩티드 또는 단백질 활성을 강화하는 방법은, 예를 들면, The method for enhancing polypeptide or protein activity using genetic engineering, for example,
1) 상기 폴리펩티드 또는 단백질을 코딩하는 유전자 또는 폴리뉴클레오티드의 세포 내 카피수 증가, 1) an increase in the intracellular copy number of a gene or polynucleotide encoding the polypeptide or protein;
2) 상기 폴리펩티드 또는 단백질을 코딩하는 염색체상의 유전자 발현조절영역을 활성이 강력한 서열로 교체하는 방법, 2) a method of replacing the gene expression control region on the chromosome encoding the polypeptide or protein with a sequence with strong activity;
3) 상기 폴리펩티드 또는 단백질의 개시코돈 또는 5'-UTR 지역의 염기서열을 변형시키는 방법, 3) a method of modifying the base sequence of the start codon or 5'-UTR region of the polypeptide or protein,
4) 상기 폴리펩티드 또는 단백질 활성이 증가되도록 염색체 상의 폴리뉴클레오티드 서열을 변형시키는 방법,4) a method of modifying a polynucleotide sequence on a chromosome to increase the polypeptide or protein activity,
5) 상기 폴리펩티드 또는 단백질의 활성을 나타내는 외래 폴리뉴클레오티드 또는 상기 폴리뉴클레오티드의 코돈 최적화된 변이형 폴리뉴클레오티드의 도입, 또는5) introduction of a foreign polynucleotide exhibiting the activity of the polypeptide or protein or a codon-optimized variant polynucleotide of the polynucleotide, or
6) 상기 방법들의 조합 등에 의하여 수행될 수 있으나, 이에 제한되지 않는다.6) It may be performed by a combination of the above methods, but is not limited thereto.
상기 단백질 공학을 이용하여 폴리펩티드 또는 단백질 활성을 강화하는 방법은, 예를 들면, 폴리펩티드 또는 단백질의 삼차구조를 분석하여 노출 부위를 선택하여 변형하거나 화학적으로 수식하는 방법 등에 의하여 수행될 수 있으나, 이에 제한되지 않는다.The method for enhancing polypeptide or protein activity using the protein engineering may be performed by, for example, a method of selecting an exposed site by analyzing the tertiary structure of the polypeptide or protein and modifying or chemically modifying it, but is limited thereto. doesn't happen
상기 1) 폴리펩티드 또는 단백질을 코딩하는 유전자 또는 폴리뉴클레오티드의 세포 내 카피수 증가는, 당업계에 알려진 임의의 방법, 예를 들면, 해당 폴리펩티드 또는 단백질을 코딩하는 유전자 또는 폴리뉴클레오티드가 작동가능하게 연결된, 숙주와 무관하게 복제되고 기능할 수 있는 벡터가 숙주세포 내에 도입됨으로써 수행될 수 있다. 또는, 상기 유전자가 작동가능하게 연결된, 숙주세포 내의 염색체 내로 상기 유전자 또는 폴리뉴클레오티드를 삽입시킬 수 있는 벡터가 숙주세포 내에 도입됨으로써 수행될 수 있으나, 이에 제한되지 않는다. 상기 벡터는 전술한 바와 같다.1) The increase in the intracellular copy number of a gene or polynucleotide encoding a polypeptide or protein is performed by any method known in the art, for example, the gene or polynucleotide encoding the polypeptide or protein is operably linked, This can be performed by introducing a vector capable of replicating and functioning independently of a host into a host cell. Alternatively, a vector capable of inserting the gene or polynucleotide into a chromosome in the host cell, to which the gene is operably linked, may be introduced into the host cell, but is not limited thereto. The vector is the same as described above.
상기 2) 폴리펩티드 또는 단백질을 코딩하는 염색체상의 유전자 발현조절영역(또는 발현조절서열)을 활성이 강력한 서열로 교체하는 방법은, 당업계에 알려진 임의의 방법, 예를 들면, 상기 발현조절영역의 활성을 더욱 강화하도록 핵산 서열을 결실, 삽입, 비보존적 또는 보존적 치환 또는 이들의 조합으로 서열상의 변이를 유도하여 수행하거나, 더욱 강한 활성을 가지는 핵산 서열로 교체함에 의하여 수행될 수 있다. 상기 발현조절영역은, 특별히 이에 제한되지 않으나 프로모터, 오퍼레이터 서열, 리보좀 결합 부위를 코딩하는 서열, 전사 및 해독의 종결을 조절하는 서열 등을 포함할 수 있다. 상기 방법은 구체적으로 본래의 프로모터 대신 강력한 이종 프로모터를 연결시키는 것일 수 있으나, 이에 제한되지 않는다.2) The method of replacing the gene expression control region (or expression control sequence) on the chromosome encoding the polypeptide or protein with a sequence with strong activity is any method known in the art, for example, the activity of the expression control region It can be carried out by inducing a mutation in the sequence by deletion, insertion, non-conservative or conservative substitution or a combination thereof to further enhance the nucleic acid sequence, or by replacing the nucleic acid sequence with a nucleic acid sequence having stronger activity. The expression control region is not particularly limited thereto, but may include a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence for regulating the termination of transcription and translation. The method may specifically be to link a strong heterologous promoter instead of the original promoter, but is not limited thereto.
진핵 생물에 대해 공지된 프로모터의 예에는 번역 신장 인자 1(TEF1), 글리세롤-3-인산 탈수소 효소 1(GPD1), 3-포스포글리세레이트 키나제 또는 다른 글리콜분해 효소, 예컨대 에놀라제, 글리세르알데히드-3-포스페이트 데히드로게나제, 헥소키나제, 피루베이트 데카르복실라제, 포스포프룩토키나제, 글루코스-6-포스페이트 이소머라제, 3-포스포글리세레이트 뮤타제, 피루베이트 키나제, 트리오스포스페이트 이소머라제, 포스포글루코스 이소머라제, 및 글루코키나제에 대한 프로모터를 포함할 수 있고, 성장 조건에 의해 제어되는 전사의 추가의 이점을 갖는 유도성 프로모터인 다른 효모 프로모터의 예에는 알콜 데히드로게나제 2, 이소시토크로뮴 C, 산 포스파타제, 질소 대사와 연관된 분해 효소, 메탈로티오네인, 글리세르알데히드-3-포스페이트 데히드로게나제, 및 말토스 및 갈락토스 이용을 담당하는 효소에 대한 프로모터가 있고, 숙주 세포가 효모인 경우 이용가능한 프로모터는 TEF1 프로모터, TEF2 프로모터, GAL10 프로모터, GAL1 프로모터, ADH1 프로모터, ADH2 프로모터, PHO5 프로모터, GAL1-10 프로모터, TDH3 프로모터(GPD 프로모터), TDH2 프로모터, TDH1 프로모터, PGK1 프로모터, PYK2 프로모터, ENO1 프로모터, ENO2 프로모터 및 TPI1 프로모터를 포함할 수 있으며, 효모 발현에 사용하기에 적합한 벡터 및 프로모터는 EP 073657에 추가로 기재되어 있으나, 이에 한정되는 것은 아니다. 또한, 효모 인핸서도 효모 프로모터와 함께 유리하게 사용될 수 있으나, 이에 제한되지 않는다. Examples of promoters known for eukaryotes include translation elongation factor 1 (TEF1), glycerol-3-phosphate dehydrogenase 1 (GPD1), 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase, glycer Aldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosphosphate Examples of other yeast promoters that are inducible promoters that may include promoters for isomerase, phosphoglucose isomerase, and glucokinase and have the added advantage of transcription controlled by growth conditions include alcohol dehydrogenase second, isocytochromium C, acid phosphatase, degradative enzymes involved in nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and promoters for enzymes responsible for maltose and galactose utilization; , When the host cell is yeast, available promoters are TEF1 promoter, TEF2 promoter, GAL10 promoter, GAL1 promoter, ADH1 promoter, ADH2 promoter, PHO5 promoter, GAL1-10 promoter, TDH3 promoter (GPD promoter), TDH2 promoter, TDH1 promoter, Vectors and promoters suitable for use in yeast expression, which may include, but are not limited to, PGK1 promoter, PYK2 promoter, ENO1 promoter, ENO2 promoter and TPI1 promoter are further described in EP 073657. In addition, yeast enhancers may also be advantageously used in conjunction with yeast promoters, but are not limited thereto.
상기 3) 폴리펩티드 또는 단백질의 개시코돈 또는 5'-UTR 지역의 염기서열을 변형시키는 방법은, 당업계에 알려진 임의의 방법, 예를 들면, 상기 폴리펩티드 또는 단백질의 내재적 개시코돈을 상기 내재적 개시코돈에 비해 폴리펩티드 또는 단백질 발현율이 더 높은 다른 개시코돈으로 치환하는 것일 수 있으나, 이에 제한되지 않는다.3) The method of modifying the base sequence of the start codon or 5'-UTR region of the polypeptide or protein is any method known in the art, for example, the endogenous start codon of the polypeptide or protein is added to the endogenous start codon. It may be substituted with another start codon having a higher expression rate of a polypeptide or protein than that, but is not limited thereto.
상기 4) 폴리펩티드 또는 단백질 활성이 증가되도록 염색체 상의 폴리뉴클레오티드 서열을 변형시키는 방법은, 당업계에 알려진 임의의 방법, 예를 들면, 상기 폴리뉴클레오티드 서열의 활성을 더욱 강화하도록 핵산 서열을 결실, 삽입, 비보존적 또는 보존적 치환 또는 이들의 조합으로 발현조절 서열상의 변이를 유도하여 수행하거나, 더욱 강한 활성을 갖도록 개량된 폴리뉴클레오티드 서열로 교체함에 의하여 수행될 수 있다. 상기 교체는 구체적으로 상동재조합에 의하여 상기 유전자를 염색체내로 삽입하는 것일 수 있으나, 이에 제한되지 않는다.4) The method of modifying a polynucleotide sequence on a chromosome to increase polypeptide or protein activity may include any method known in the art, for example, deleting, inserting, or inserting a nucleic acid sequence to further enhance the activity of the polynucleotide sequence; It can be carried out by inducing a mutation in the expression control sequence by non-conservative or conservative substitution or a combination thereof, or by replacing it with an improved polynucleotide sequence to have stronger activity. The replacement may specifically be to insert the gene into the chromosome by homologous recombination, but is not limited thereto.
이때 사용되는 벡터는 염색체 삽입 여부를 확인하기 위한 선별 마커 (selection marker)를 추가로 포함할 수 있다. 상기 선별 마커는 전술한 바와 같다.In this case, the vector used may further include a selection marker for confirming whether or not the chromosome is inserted. The selection marker is the same as described above.
상기 5) 폴리펩티드 또는 단백질의 활성을 나타내는 외래 폴리뉴클레오티드의 도입은, 당업계에 알려진 임의의 방법, 예를 들면, 상기 폴리펩티드 또는 단백질과 동일/유사한 활성을 나타내는 폴리펩티드 또는 단백질을 코딩하는 외래 폴리뉴클레오티드, 또는 이의 코돈 최적화된 변이형 폴리뉴클레오티드를 숙주세포 내로 도입하여 수행될 수 있다. 상기 외래 폴리뉴클레오티드는 상기 폴리펩티드 또는 단백질과 동일/유사한 활성을 나타내는 한 그 유래나 서열에 제한 없이 사용될 수 있다. 또한 도입된 상기 외래 폴리뉴클레오티드가 숙주세포 내에서 최적화된 전사, 번역이 이루어지도록 이의 코돈을 최적화하여 숙주세포 내로 도입할 수 있다. 상기 도입은 공지된 형질전환 방법을 당업자가 적절히 선택하여 수행될 수 있으며, 숙주 세포 내에서 상기 도입된 폴리뉴클레오티드가 발현됨으로써 폴리펩티드 또는 단백질이 생성되어 그 활성이 증가될 수 있다.5) The introduction of a foreign polynucleotide exhibiting the activity of a polypeptide or protein can be carried out by any method known in the art, for example, a foreign polynucleotide encoding a polypeptide or protein exhibiting the same/similar activity as the polypeptide or protein; Or it may be carried out by introducing a codon-optimized variant polynucleotide thereof into a host cell. The foreign polynucleotide may be used without limitation in origin or sequence as long as it exhibits the same/similar activity as the polypeptide or protein. In addition, the introduced foreign polynucleotide can be introduced into the host cell by optimizing its codon so that the optimized transcription and translation are performed in the host cell. The introduction can be carried out by appropriately selecting a known transformation method by those skilled in the art, and by expressing the introduced polynucleotide in a host cell, a polypeptide or protein is produced and its activity can be increased.
마지막으로, 6) 상기 방법들의 조합은 상기 1) 내지 5) 중 어느 하나 이상의 방법을 함께 적용하여 수행될 수 있다.Finally, 6) the combination of the methods may be performed by applying any one or more methods of 1) to 5) together.
이와 같은 폴리펩티드 또는 단백질 활성의 강화는, 상응하는 폴리펩티드 또는 단백질의 활성 또는 농도가 야생형이나 변형 전 미생물 균주에서 발현된 폴리펩티드 또는 단백질의 활성 또는 농도를 기준으로 하여 증가되거나, 해당 폴리펩티드 또는 단백질로부터 생산되는 산물의 양이 증가되는 것일 수 있으나, 이에 제한되는 것은 아니다.The enhancement of such polypeptide or protein activity is a function in which the activity or concentration of the corresponding polypeptide or protein is increased relative to the activity or concentration of the polypeptide or protein expressed in the wild-type or pre-modified microbial strain, or is produced from the polypeptide or protein. The amount of the product may be increased, but is not limited thereto.
본 출원에서 용어, "변형 전 균주" 또는 “변형 전 미생물”은 미생물에 자연적으로 발생할 수 있는 돌연변이를 포함하는 균주를 제외하는 것이 아니며, 야생형 균주 또는 천연형 균주 자체이거나, 자연적 또는 인위적 요인에 의한 유전적 변이로 형질이 변화되기 전 균주를 의미할 수 있다. 상기 “변형 전 균주” 또는 “변형 전 미생물”은 “비변이 균주”, “비변형 균주”, “비변이 미생물”, “비변형 미생물” 또는 “기준 미생물”과 혼용될 수 있다.In the present application, the term "pre-transformation strain" or "pre-transformation microorganism" does not exclude a strain containing a mutation that can occur naturally in a microorganism, it is a wild-type strain or a natural-type strain itself, or caused by natural or artificial factors It may refer to a strain before the trait is changed due to genetic mutation. The “pre-modified strain” or “pre-modified microorganism” may be used interchangeably with “unmodified strain”, “unmodified strain”, “unmodified microorganism”, “unmodified microorganism” or “reference microorganism”.
본 출원에서 상기 글루타메이트-시스테인 리가아제 변이체를 포함하거나 이를 코딩하는 폴리뉴클레오티드, 또는 상기 폴리뉴클레오티드를 포함하는 벡터를 포함하는 미생물은, 재조합 미생물일 수 있고, 상기 재조합은 형질전환과 같은 유전적 변형(genetically modification)에 의해 이루어질 수 있다.In the present application, a microorganism comprising a polynucleotide comprising or encoding the glutamate-cysteine ligase variant, or a vector comprising the polynucleotide may be a recombinant microorganism, and the recombination is genetic modification such as transformation ( genetically modified).
예를 들면, 상기 폴리뉴클레오티드를 포함하는 벡터로 형질전환에 의해 제조되는 재조합 미생물일 수 있으나, 이에 제한되지 않는다. 상기 재조합 미생물은 효모일 수 있으며, 그 예로, 사카로마이세스 속 미생물일 수 있고, 구체적으로 사카로마이세스 세레비지애(Saccharomyces cerevisiae)일 수 있으나, 이에 제한되지 않는다. For example, it may be a recombinant microorganism prepared by transformation with a vector containing the polynucleotide, but is not limited thereto. The recombinant microorganism may be yeast, for example, may be a microorganism of the genus Saccharomyces, specifically Saccharomyces cerevisiae , but may be, but is not limited thereto.
본 출원의 다른 하나의 양태는 상기 미생물을 배양하는 단계를 포함하는 글루타치온 제조방법을 제공한다. 상기 미생물, 글루타치온에 대해서는 전술한 바와 같다. Another aspect of the present application provides a glutathione production method comprising the step of culturing the microorganism. The microorganism, glutathione, is the same as described above.
본 출원의 균주의 배양에 사용되는 배지 및 기타 배양 조건은 통상의 사카로마이세스속 미생물의 배양에 사용되는 배지라면 특별한 제한 없이 어느 것이나 사용될 수 있으며, 구체적으로는 본 출원의 균주를 적당한 탄소원, 질소원, 인원, 무기화합물, 아미노산 및/또는 비타민 등을 함유한 통상의 배지 내에서 호기성 또는 혐기성 조건 하에서 온도, pH 등을 조절하면서 배양할 수 있다.Any medium and other culture conditions used for culturing the strain of the present application may be used without any particular limitation as long as it is a medium used for culturing a microorganism of the genus Saccharomyces, specifically, the strain of the present application is selected from a suitable carbon source; It can be cultured while controlling temperature, pH, etc. under aerobic or anaerobic conditions in a normal medium containing a nitrogen source, phosphorus, inorganic compounds, amino acids and/or vitamins.
본 출원에서 상기 탄소원으로는 글루코오스, 프룩토오스, 수크로오스, 말토오스 등과 같은 탄수화물; 만니톨, 소르비톨 등과 같은 당 알코올, 피루브산, 락트산, 시트르산 등과 같은 유기산; 글루타메이트, 메티오닌, 라이신 등과 같은 아미노산 등이 포함될 수 있으나, 이에 제한되지 않는다. 또한, 전분 가수분해물, 당밀, 블랙스트랩 당밀, 쌀겨울, 카사버, 사탕수수 찌꺼기 및 옥수수 침지액 같은 천연의 유기 영양원을 사용할 수 있으며, 글루코오스 및 살균된 전처리 당밀(즉, 환원당으로 전환된 당밀) 등과 같은 탄수화물이 사용될 수 있고, 그 외의 적정량의 탄소원을 제한 없이 다양하게 이용할 수 있다. 이들 탄소원은 단독으로 사용되거나 2 종 이상이 조합되어 사용될 수 있다.As the carbon source in the present application, carbohydrates such as glucose, fructose, sucrose, maltose; sugar alcohols such as mannitol and sorbitol; organic acids such as pyruvic acid, lactic acid, citric acid and the like; Amino acids such as glutamate, methionine, lysine and the like may be included, but are not limited thereto. In addition, natural organic nutrient sources such as starch hydrolyzate, molasses, blackstrap molasses, rice winter, cassava, sugarcane offal and corn steep liquor can be used, including glucose and pasteurized pretreated molasses (i.e. molasses converted to reducing sugar). Carbohydrates such as, etc. may be used, and other appropriate amounts of carbon sources may be variously used without limitation. These carbon sources may be used alone or in combination of two or more.
상기 질소원으로는 암모니아, 황산암모늄, 염화암모늄, 초산암모늄, 인산암모늄, 탄산안모늄, 질산암모늄 등과 같은 무기질소원; 아미노산, 펩톤, NZ-아민, 육류 추출물, 효모 추출물, 맥아 추출물, 옥수수 침지액, 카세인 가수분해물, 어류 또는 그의 분해생성물, 탈지 대두 케이크 또는 그의 분해 생성물 등과 같은 유기 질소원이 사용될 수 있다. 이들 질소원은 단독으로 사용되거나 2 종 이상이 조합되어 사용될 수 있으나, 이에 제한되지 않는다.Examples of the nitrogen source include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Organic nitrogen sources such as amino acids, peptones, NZ-amines, meat extracts, yeast extracts, malt extracts, corn steep liquor, casein hydrolysates, fish or degradation products thereof, defatted soybean cakes or degradation products thereof and the like can be used. These nitrogen sources may be used alone or in combination of two or more, but is not limited thereto.
상기 인원으로는 인산 제1칼륨, 인산 제2칼륨, 또는 이에 대응되는 소디움-함유 염 등이 포함될 수 있다. 무기화합물로는 염화나트륨, 염화칼슘, 염화철, 황산마그네슘, 황산철, 황산망간, 탄산칼슘 등이 사용될 수 있다. The phosphorus may include potassium monobasic phosphate, dipotassium phosphate, or a sodium-containing salt corresponding thereto. As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. may be used.
그 외에 상기 배지에는 아미노산, 비타민 및/또는 적절한 전구체 등이 포함될 수 있다. 구체적으로, 상기 균주의 배양 배지에는 L-아미노산 등이 첨가될 수 있다. 구체적으로는 글리신(glycine), 글루타메이트(glutamate), 및/또는 시스테인(cysteine) 등이 첨가될 수 있고, 필요에 따라서는 라이신(lysine) 등의 L-아미노산 이 더 첨가될 수 있으나 반드시 이에 제한되지 않는다. In addition, the medium may contain amino acids, vitamins and/or appropriate precursors. Specifically, L-amino acid and the like may be added to the culture medium of the strain. Specifically, glycine, glutamate, and/or cysteine may be added, and if necessary, L-amino acids such as lysine may be further added, but not necessarily limited thereto. does not
상기 배지 또는 전구체는 배양물에 회분식 또는 연속식으로 첨가될 수 있으며, 이에 제한되지 않는다.The medium or precursor may be added to the culture in a batch or continuous manner, but is not limited thereto.
본 출원에서, 균주의 배양 중에 수산화암모늄, 수산화칼륨, 암모니아, 인산, 황산 등과 같은 화합물을 배양물에 적절한 방식으로 첨가하여, 배양물의 pH를 조정할 수 있다. 또한, 배양 중에는 지방산 폴리글리콜 에스테르와 같은 소포제를 사용하여 기포 생성을 억제할 수 있다. 또한, 배양물의 호기 상태를 유지하기 위하여, 배양물 내로 산소 또는 산소 함유 기체를 주입하거나 혐기 및 미호기 상태를 유지하기 위해 기체의 주입 없이 혹은 질소, 수소 또는 이산화탄소 가스를 주입할 수 있다.In the present application, by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, sulfuric acid, etc. to the culture in an appropriate manner during the culture of the strain, the pH of the culture can be adjusted. In addition, during culturing, an antifoaming agent such as fatty acid polyglycol ester may be used to suppress bubble formation. In addition, in order to maintain the aerobic state of the culture, oxygen or oxygen-containing gas may be injected into the culture, or nitrogen, hydrogen or carbon dioxide gas may be injected with or without gas to maintain anaerobic and microaerobic conditions.
배양물의 온도는 25℃내지 40℃일 수 있으며, 보다 구체적으로는 28℃내지 37℃일 수 있으나 이에 제한되지 않는다. 배양 기간은 원하는 유용 물질의 생성량이 수득될 때까지 계속될 수 있으며, 구체적으로는 1 시간 내지 100 시간일 수 있으나 이에 제한되지 않는다.The temperature of the culture may be 25°C to 40°C, and more specifically, 28°C to 37°C, but is not limited thereto. The incubation period may be continued until a desired production amount of useful substances is obtained, and specifically, it may be 1 hour to 100 hours, but is not limited thereto.
상기 글루타치온 제조방법은, 상기 배양 단계 이후, 추가적인 공정을 더 포함할 수 있다. 상기 추가 공정은 글루타치온 용도에 따라 적절히 선택될 수 있다.The glutathione production method may further include an additional process after the culturing step. The additional process may be appropriately selected depending on the use of glutathione.
구체적으로, 상기 글루타치온 제조방법은 상기 배양 단계에 의해 균체 내에 축적된 글루타치온을 회수하는 단계를 포함할 수 있고, 예를 들면 상기 배양 단계 이후 상기 균주, 이의 건조물, 추출물, 배양물, 파쇄물 중에서 선택된 하나 이상의 물질로부터 글루타치온을 회수하는 단계를 포함할 수 있다.Specifically, the glutathione production method may include the step of recovering glutathione accumulated in the cells by the culturing step, for example, after the culturing step, one selected from the strain, its dried product, extract, culture, and lysate. It may include the step of recovering glutathione from the above substances.
상기 방법은 상기 회수 단계 이전, 혹은 동시에 균주를 용균시키는 단계를 추가로 포함할 수 있다. 균주의 용균은 본 출원이 속하는 기술 분야에서 통상적으로 사용되는 방법, 예를 들어, 용균용 완충용액, 소니케이터, 열 처리 및 후렌치 프레서 등에 의해 실시할 수 있다. 또한 상기 용균 단계는 세포벽 분해 효소, 핵산 분해 효소, 핵산 전이 효소, 단백질 분해 효소 등 효소반응을 포함할 수 있으나 이에 제한되지 않는다.The method may further include a step of lysing the strain before or simultaneously with the recovery step. The lysis of the strain may be carried out by a method commonly used in the technical field to which the present application belongs, for example, a lysis buffer solution, a sonicator, heat treatment, and a French presser. In addition, the lysis step may include an enzymatic reaction such as a cell wall degrading enzyme, a nucleic acid degrading enzyme, a nucleic acid transfer enzyme, a proteolytic enzyme, but is not limited thereto.
본 출원의 목적상, 상기 글루타치온 제조방법을 통해 글루타치온이 고함량으로 포함된 건조 효모(Dry yeast), 효모 추출물(yeast extract), 효모추출물 혼합 분말(yeast extract mix powder), 순수 정제된 글루타치온(pure glutathione)을 제조할 수 있으나 이에 제한되지 않으며, 목적하는 제품에 따라 적절하게 제조될 수 있다.For the purpose of the present application, dry yeast containing a high content of glutathione through the glutathione manufacturing method, yeast extract, yeast extract mix powder, pure purified glutathione (pure) glutathione), but is not limited thereto, and may be appropriately manufactured according to the desired product.
본 출원에서 건조 효모(dry yeast)는 "균주 건조물" 등의 용어와 교환적으로 사용될 수 있다. 상기 건조 효모는 글루타치온을 축적한 효모 균체를 건조시켜 제조할 수 있으며, 구체적으로 사료용 조성물, 식품용 조성물 등에 포함될 수 있으나, 이에 제한되지 않는다.In the present application, dry yeast may be used interchangeably with terms such as "dried strain". The dry yeast may be prepared by drying yeast cells accumulating glutathione, and may be specifically included in a composition for feed, a composition for food, and the like, but is not limited thereto.
본 출원에서 효모 추출물(yeast extract)은 "균주 추출물" 등의 용어와 상호 교환적으로 사용될 수 있다. 상기 균주 추출물은, 상기 균주의 균체에서 세포벽을 분리하고 남은 물질을 의미할 수 있다. 구체적으로, 균체를 용균시켜 수득한 성분에서 세포벽을 제외한 나머지 성분을 의미할 수 있다. 상기 균주 추출물은 글루타치온을 포함하며, 글루타치온 외의 성분으로는 단백질, 탄수화물, 핵산, 섬유질 중 하나 이상의 성분이 포함되어 있을 수 있으나 이에 제한되지 않는다. In the present application, yeast extract may be used interchangeably with terms such as "strain extract". The strain extract may mean a material remaining after separating the cell wall from the cells of the strain. Specifically, it may refer to the remaining components excluding the cell wall from the components obtained by lysing the cells. The extract of the strain includes glutathione, and components other than glutathione may include at least one of protein, carbohydrate, nucleic acid, and fiber, but is not limited thereto.
상기 회수 단계는 당해 기술 분야에 공지된 적합한 방법을 이용하여, 목적 물질인 글루타치온을 회수할 수 있다.In the recovery step, glutathione, a target material, may be recovered using a suitable method known in the art.
상기 회수 단계는 정제 공정을 포함할 수 있다. 상기 정제 공정은 균주로부터 글루타치온만을 분리하여 순수 정제하는 것일 수 있다. 상기 정제 공정을 통해, 순수 정제된 글루타치온(pure glutathione)이 제조될 수 있다.The recovery step may include a purification process. The purification process may be pure purification by separating only glutathione from the strain. Through the purification process, pure purified glutathione may be prepared.
필요에 따라, 상기 글루타치온 제조방법은, 상기 배양 단계 이후 수득된 균주, 이의 건조물, 추출물, 배양물, 파쇄물 및 이들로부터 회수된 글루타치온 중에서 선택된 물질과 부형제를 혼합하는 단계를 더 포함할 수 있다. 상기 혼합 단계를 통해 효모추출물 혼합 분말(yeast extract mix powder)이 제조될 수 있다.If necessary, the method for preparing glutathione may further include a step of mixing a material selected from among the strains obtained after the culturing step, a dried product, an extract, a culture, a lysate, and glutathione recovered therefrom and an excipient. Through the mixing step, yeast extract mix powder can be prepared.
상기 부형제는 목적하는 용도나 형태에 따라 적절히 선택하여 사용할 수 있으며, 예를 들어, 전분, 글루코오스, 셀룰로오스, 락토오스, 글리코겐, D-만니톨, 소르비톨, 락티톨, 말토덱스트린, 탄산칼슘, 합성규산알루미늄, 인산일수소칼슘, 황산칼슘, 염화나트륨, 탄산수소나트륨, 정제 라놀린, 덱스트린, 알긴산나트륨, 메틸셀룰로오스, 콜로이드성실리카겔, 하이드록시프로필스타치, 하이드록시프로필메틸셀루로오스, 프로필렌글리콜, 카제인, 젖산칼슘, 프리모젤, 아라비아 검 중에서 선택되는 것일 수 있으며, 구체적으로는 전분, 글루코오스, 셀룰로오스, 락토오스, 덱스트린, 글리코겐, D-만니톨, 말토덱스트린 중에서 선택되는 하나 이상의 성분일 수 있으나 이에 제한되지 않는다.The excipient may be appropriately selected and used according to the intended use or form, for example, starch, glucose, cellulose, lactose, glycogen, D-mannitol, sorbitol, lactitol, maltodextrin, calcium carbonate, synthetic aluminum silicate, Calcium monohydrogen phosphate, calcium sulfate, sodium chloride, sodium hydrogen carbonate, purified lanolin, dextrin, sodium alginate, methylcellulose, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethylcellulose, propylene glycol, casein, calcium lactate , Primogel, and gum arabic, specifically, starch, glucose, cellulose, lactose, dextrin, glycogen, D-mannitol, may be one or more components selected from maltodextrin, but is not limited thereto.
상기 부형제는, 예를 들어, 보존제, 습윤제, 분산제, 현탁화제, 완충제, 안정화제 또는 등장화제 등을 포함할 수 있으나, 이에 한정되는 것은 아니다.The excipient may include, for example, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizing agent, or an isotonic agent, but is not limited thereto.
본 출원의 다른 하나의 양태는 본 출원의 변이체의 글루타치온 생산용도를 제공한다. Another aspect of the present application provides a glutathione production use of the variant of the present application.
본 출원의 하나의 양태는 본출원의 변이체 글루타메이트-시스테인 리가아제 변이체를 포함하는 미생물의 글루타치온 생산 용도를 제공한다. One aspect of the present application provides a glutathione production use of a microorganism comprising the mutant glutamate-cysteine ligase variant of the present application.
상기 변이체, 폴리튜클레오티드, 미생물에 대해서는 전술한 바와 같다. The variants, polynucleotides, and microorganisms are the same as described above.
이하 본 출원을 실시예 및 실험예를 통하여 보다 상세하게 설명한다. 그러나 이들 실시예 및 실험예는 본 출원을 예시적으로 설명하기 위한 것으로 본 출원의 범위가 이들 실시예 및 실험예에 한정되는 것은 아니다.Hereinafter, the present application will be described in more detail through Examples and Experimental Examples. However, these Examples and Experimental Examples are for illustrative purposes of the present application, and the scope of the present application is not limited to these Examples and Experimental Examples.
실시예 1: 글루타치온 생산 균주 선별 및 개량Example 1: Selection and improvement of glutathione-producing strains
실시예 1-1: 글루타치온 생산균주 선별Example 1-1: Selection of glutathione-producing strains
다양한 균주를 함유하고 있는 누룩으로부터 균주를 수득하고 이를 개량하여 글루타치온 생산능을 갖는 균주를 선별하였다.A strain having a glutathione-producing ability was selected by obtaining a strain from yeast containing various strains and improving it.
구체적으로, 대한민국 경기도 용인 이천 평택 화성 지역 등 총 20개 지역에서 쌀, 보리, 녹두, 귀리 등 곡물시료를 채취하여 분쇄한 후 반죽하여 헝겊에 싸고 단단히 눌러 모양을 만든 다음, 짚으로 싸서 10일간 발효시킨 후, 서서히 건조시켜서 누룩을 제조하였다. Specifically, grain samples such as rice, barley, mung bean, and oats are collected from a total of 20 regions including Yongin, Icheon, Pyeongtaek, and Hwaseong areas in Gyeonggi-do, Korea, and then kneaded, wrapped in a cloth, pressed firmly to form a shape, and then wrapped with straw and fermented for 10 days. After that, it was dried slowly to prepare yeast.
제조된 누룩으로부터 다양한 균주를 분리하고자 하기와 같이 실험을 수행하였다. 5g의 누룩에 45 ml의 식염수를 첨가하여 혼합기로 분쇄하였다. 효모 균주의 순수분리를 위해 serial dilution하여 YPD Agar(Yeast extract 10 g/L, Bacto peptone 20 g/L, Glucose 20 g/L, 증류수 1리터 기준)에 spreading하여 30℃에서 48시간 배양하였다. 그리고, colony 형태와 현미경 검증을 통해 효모의 colony를 YPD agar에 streaking 하였다. 250 ml 삼각플라스크에 YPD broth를 25ml 분주하고 순수 분리된 균주를 접종하여 48시간동안 진탕 배양(30℃, 200 rpm)하여 글루타치온 생산량을 확인하여 균주 스크리닝을 수행하였다. Experiments were performed as follows to isolate various strains from the prepared yeast. 45 ml of brine was added to 5 g of koji and pulverized with a mixer. For pure separation of yeast strains, serial dilution was carried out, spread on YPD Agar (Yeast extract 10 g/L, Bacto peptone 20 g/L, Glucose 20 g/L, distilled water based on 1 liter), and cultured at 30° C. for 48 hours. Then, yeast colonies were streaked on YPD agar through colony morphology and microscopic verification. 25ml of YPD broth was dispensed in a 250ml Erlenmeyer flask, inoculated with the pure isolated strain, and cultured with shaking (30℃, 200rpm) for 48 hours to check the glutathione production, and strain screening was performed.
1차적으로 분리된 균주들의 개량을 위해, 분리된 균주에 돌연변이(random mutation)를 유도하였다. 구체적으로 상기 누룩으로부터 분리한 효모중 글루타치온 생산이 확인된 균주를 분리하여 CJ-37 균주로 명명하였다. CJ-37 균주를 고체배지에 배양한 후 broth에 접종하여 배양액을 수득하였으며, UV 램프를 이용하여 균체에 UV를 조사하였다. 이후, UV 조사된 배양액을 평판배지에 도말하여 콜로니를 형성한 변이 균주만을 분리 수득하였으며, 이들의 글루타치온 생산량을 확인하였다.For the improvement of the primary isolated strains, a mutation (random mutation) was induced in the isolated strain. Specifically, a strain in which glutathione production was confirmed among the yeast isolated from the yeast was isolated and named CJ-37 strain. After culturing the CJ-37 strain on a solid medium, the broth was inoculated to obtain a culture solution, and UV was irradiated to the cells using a UV lamp. Thereafter, only mutant strains that formed colonies were isolated and obtained by smearing the UV-irradiated culture medium on a plate medium, and their glutathione production was confirmed.
그 결과, 변이 균주 중 가장 우수한 글루타치온 생산량을 나타내는 균주를 글루타치온 생산 균주로 선별하여 CJ-5 균주로 명명하였으며, 부다페스트 조약 하의 국제기탁기관인 한국미생물보존센터(Korean CultureCenter of Microorganisms, KCCM)에 2019년 7월 31일자로 기탁하여 기탁번호 KCCM12568P를 부여 받았다.As a result, the strain showing the best glutathione production among the mutated strains was selected as the glutathione-producing strain and named CJ-5 strain. It was deposited on the 31st of the month and given the deposit number KCCM12568P.
실시예 1-2: 글루타치온 생산능 증가를 위한 추가 개량 실험 Example 1-2: Additional improvement experiment for increasing glutathione production capacity
CJ-5 균주의 글루타치온 생산능을 추가적으로 개선하기 위해 다음과 같은 방법으로 돌연변이를 유도하였다. In order to further improve the glutathione-producing ability of the CJ-5 strain, mutations were induced in the following way.
CJ-5 균주를 고체배지에 배양한 후 broth에 접종하여 배양액을 수득하였으며, UV 램프를 이용하여 균체에 UV를 조사하였다. 이후, UV 조사된 배양액을 평판배지에 도말하여 콜로니를 형성한 변이 균주만을 분리 수득하였으며, 글루타치온 생산능이 가장 많이 향상된 균주를 분리하여 CC02-2490 균주로 명명하고 부다페스트 조약 하의 국제기탁기관인 한국미생물보존센터(Korean CultureCenter of Microorganisms, KCCM) 에 2020년 1월 17일자로 기탁하여 수탁번호 KCCM12659P를 부여받았다. After culturing the CJ-5 strain on a solid medium, the broth was inoculated to obtain a culture solution, and UV was irradiated to the cells using a UV lamp. After that, only the mutant strains that formed colonies were isolated and obtained by smearing the UV-irradiated culture medium on a plate medium, and the strain with the most improved glutathione production ability was isolated and named as the CC02-2490 strain, and the Korea Microorganism Conservation Center, an international depository under the Budapest Treaty. It was deposited with the (Korean Culture Center of Microorganisms, KCCM) on January 17, 2020 and was given an accession number KCCM12659P.
상기 CC02-2490 균주의 글루타치온 생산능 증가와 관련하여 글루타치온 생합성유전자 gsh1의 염기서열을 분석한 결과, gsh1 유전자가 코딩하는 GSH1 단백질(서열번호 1)의 86번째 아미노산인 시스테인이 아르기닌으로 치환되었음을 확인하였다. As a result of analyzing the nucleotide sequence of the glutathione biosynthesis gene gsh1 in relation to the increase in the glutathione production capacity of the CC02-2490 strain, it was confirmed that the 86th amino acid cysteine of the GSH1 protein (SEQ ID NO: 1) encoded by the gsh1 gene was substituted with arginine. .
실시예 1-3: 글루타치온 생산능 증가를 위한 추가 개량 실험 Example 1-3: Additional improvement experiment for increasing glutathione production capacity
CC02-2490 균주의 글루타치온 생산능을 추가적으로 개선하기 위해 다음과 같은 방법으로 돌연변이를 유도하였다. In order to further improve the glutathione-producing ability of the CC02-2490 strain, mutations were induced in the following way.
CC02-2490 균주를 고체배지에 배양한 후 broth에 접종하여 배양액을 수득하였으며, UV 램프를 이용하여 균체에 UV를 조사하였다. 이후, UV 조사된 배양액을 평판배지에 도말하여 콜로니를 형성한 변이 균주만을 분리 수득하였으며, 글루타치온 생산능이 가장 많이 향상된 균주를 분리하여 CC02-2544 균주로 명명하고 부다페스트 조약 하의 국제기탁기관인 한국미생물보존센터(Korean CultureCenter of Microorganisms, KCCM) 에 2020년 2월 20일자로 기탁하여 수탁번호 KCCM12674P를 부여받았다. After culturing the CC02-2490 strain on a solid medium, the broth was inoculated to obtain a culture solution, and UV was irradiated to the cells using a UV lamp. After that, only the mutant strains that formed colonies were isolated and obtained by smearing the UV-irradiated culture medium on a plate medium, and the strain with the most improved glutathione production ability was isolated and named as the CC02-2544 strain. It was deposited with the (Korean Culture Center of Microorganisms, KCCM) on February 20, 2020 and was given an accession number KCCM12674P.
상기 CC02-2544 균주의 글루타치온 생산능 증가와 관련하여 글루타치온 생합성유전자 gsh1의 염기서열을 분석한 결과, GSH1 ORF 상단 -250(C→T), -252(G→A), -398(A→T), -399(A→C), -407(T→C), -409(T→C) 위치에서 변이가 일어났음을 확인하였다(서열번호 12). As a result of analyzing the nucleotide sequence of the glutathione biosynthesis gene gsh1 in relation to the increase in the glutathione production capacity of the CC02-2544 strain, the upper end of the GSH1 ORF -250 (C → T), -252 (G → A), -398 (A → T) ), -399 (A→C), -407 (T→C), -409 (T→C), it was confirmed that mutations occurred at positions (SEQ ID NO: 12).
실시예 2: 글루타치온 생산능 증가를 위한 CC02-2544 균주의 추가 개량 실험Example 2: Additional improvement experiment of strain CC02-2544 for increasing glutathione production capacity
CC02-2544 균주의 글루타치온 생산능을 추가적으로 개선하기 위해 다음과 같은 방법으로 돌연변이를 유도하였다. In order to further improve the glutathione-producing ability of the CC02-2544 strain, mutations were induced in the following way.
CC02-2544 균주를 고체배지에 배양한 후 broth에 접종하여 배양액을 수득하였으며, UV 램프를 이용하여 균체에 UV를 조사하였다. 이후, UV 조사된 배양액을 평판배지에 도말하여 콜로니를 형성한 변이 균주만을 분리 수득하여 염기서열을 분석하였다.After culturing the CC02-2544 strain on a solid medium, the broth was inoculated to obtain a culture solution, and UV was irradiated to the cells using a UV lamp. Thereafter, only mutant strains that formed colonies by spreading the UV-irradiated culture medium on a plate medium were isolated and obtained and the base sequence was analyzed.
실험 결과, 글루타치온 함량이 27% 향상된 균주의 글루타치온 생합성 유전자 gsh1이 코딩하는 단백질인 GSH1의 653번째 아미노산(글리신)이 메티오닌으로 치환된 것을 확인하였다. 이 균주를 CC02-2816으로 명명하고 부다페스트조약 하의 수탁기관인 한국미생물보존센터에 2020년 12월 8일자로 기탁하여 수탁번호 KCCM12891P를 부여받았다.As a result of the experiment, it was confirmed that the amino acid at position 653 (glycine) of GSH1, which is a protein encoded by the glutathione biosynthesis gene gsh1, of a strain having a glutathione content improved by 27% was substituted with methionine. This strain was named CC02-2816 and deposited with the Korea Microorganism Conservation Center, a trustee institution under the Budapest Treaty, on December 8, 2020, and was given accession number KCCM12891P.
실시예 3: GSH1 G653 잔기 변이 실험Example 3: GSH1 G653 residue mutation experiment
상기 실시예 2의 결과로부터 GSH1 단백질의 653번 위치가 글루타치온 생산에 중요할 것이라고 판단하여, GSH1 단백질의 653번째 아미노산이 다른 아미노산으로 치환된 변이 단백질을 발현하도록 사카로마이세스 세레비지애(S. cerevisiae) CEN.PK2-1D 및 CC02-2544 균주의 변이 균주를 제작하여 글루타치온 생산량 증가 여부를 확인하고자 하였다. 한편 앞서 언급한 바와 같이, CC02-2544 균주의 경우는 GSH1 C86R변이주에 GSH1 ORF 상단 -250(C→T), -252(G→A), -398(A→T), -399(A→C), -407(T→C), -409(T→C) 위치에 변이가 있는 균주이며 해당 균주에는 GSH1 단백질의 653번째 아미노산의 변이를 추가적으로 도입하였다.From the results of Example 2, it was determined that the 653 position of the GSH1 protein would be important for glutathione production, and S. cerevisiae (S. cerevisiae) CEN.PK2-1D and CC02-2544 strains were prepared to determine whether glutathione production was increased. Meanwhile, as mentioned above, in the case of the CC02-2544 strain, the GSH1 ORF upper end -250(C→T), -252(G→A), -398(A→T), -399(A→ C), -407 (T→C), -409 (T→C) is a strain with mutations, and a mutation of amino acid 653 of the GSH1 protein was additionally introduced into the strain.
사카로마이세스 세레비지애 효모의 GSH1 단백질의 653번 아미노산을 메티오닌으로 치환한 균주를 제작하기 위하여 Lee TH, et al.(J. Microbiol. Biotechnol. (2006), 16(6), 979-982) 논문에 개시된 내용을 참고로 하여 pWAL100 및 pWBR100 플라스미드를 이용하였다. 구체적으로 CJ-5 균주의 genomic DNA를 주형(template)으로 하여 다음과 같이 PCR을 수행하였다. 서열번호 4와 서열번호 5의 프라이머를 이용한 PCR을 수행하여 N-terminal BamHI flanking 서열, GSH1 ORF의 개시코돈 및 G653M 변이 암호화 서열을 포함하는 GSH1 N-terminal 일부 서열을 확보하고, 서열번호 6와 서열번호 7의 프라이머를 이용하여 C-terminal XhoI flanking 서열, GSH1 ORF 종결코돈 및 G653M 변이 암호화 서열을 포함하는 GSH1 C-terminal 일부 서열을 확보하였다. 이후 이 두 서열을 주형(template)으로 하여 서열번호 4와 서열번호 7을 이용하여 overlap PCR을 수행한 결과 653번째 아미노산이 메티오닌으로 치환된 GSH1 변이 단백질 암호화 서열, N-terminal BamHI, C-terminal XhoI 제한효소 서열을 포함하는 GSH1 ORF 절편을 확보하였다. 상기 ORF 절편은 BamHI 및 XhoI처리 후 동일한 효소로 처리한 pWAL100 벡터에 클로닝하여 pWAL100-GSH1(G653M)벡터를 제조하였다.In order to prepare a strain in which amino acid 653 of the Saccharomyces cerevisiae yeast GSH1 protein was substituted with methionine, Lee TH, et al. (J. Microbiol. Biotechnol. (2006), 16(6), 979-982) ) The pWAL100 and pWBR100 plasmids were used with reference to the contents disclosed in the paper. Specifically, PCR was performed as follows using the genomic DNA of the CJ-5 strain as a template. PCR was performed using the primers of SEQ ID NO: 4 and SEQ ID NO: 5 to secure a partial sequence of GSH1 N-terminal including the N-terminal BamHI flanking sequence, the start codon of GSH1 ORF, and the G653M variant coding sequence, and SEQ ID NO: 6 and the sequence A partial sequence of GSH1 C-terminal including the C-terminal XhoI flanking sequence, the GSH1 ORF stop codon and the G653M mutation coding sequence was obtained by using the primer of No. 7. Then, as a result of performing overlap PCR using SEQ ID NO: 4 and SEQ ID NO: 7 using these two sequences as a template, the GSH1 mutant protein coding sequence in which the 653th amino acid was substituted with methionine, N-terminal BamHI, C-terminal XhoI A GSH1 ORF fragment containing a restriction enzyme sequence was obtained. The ORF fragment was cloned into the pWAL100 vector treated with the same enzyme after treatment with BamHI and XhoI to prepare a pWAL100-GSH1(G653M) vector.
또한, CJ-5 균주의 genomic DNA를 template으로 하여 서열번호 8과 서열번호 9를 이용한 PCR을 수행하여 N-terminal SpeI, C-terminal NcoI 제한효소 서열을 포함하는 GSH1 ORF 종결코돈 이후 500bp 단편을 확보하고 SpeI, NcoI 제한효소를 처리하였다. 이후 동일한 제한효소를 처리한 pWBR100에 클로닝하여 pWBR100-GSH1벡터를 제조하였다.In addition, PCR using SEQ ID NO: 8 and SEQ ID NO: 9 using the genomic DNA of the CJ-5 strain as a template was performed to secure a 500 bp fragment after the GSH1 ORF stop codon including the N-terminal SpeI and C-terminal NcoI restriction enzyme sequences and SpeI and NcoI restriction enzymes were treated. After cloning into pWBR100 treated with the same restriction enzyme, pWBR100-GSH1 vector was prepared.
최종적으로 효모에 도입할 DNA 절편을 제작하기 위해 앞서 제작한 pWAL100-GSH1(G653M) 벡터를 주형(template)으로 하여 서열번호 4와 서열번호 10의 프라이머를 이용하여 메티오닌 변이 암호화서열과 KlURA3 일부를 포함하는 PCR 산물을 획득하고 pWBR100-GSH1벡터를 주형(template)으로 하여 서열번호 11과 서열번호 9의 프라이머를 이용하여 KlURA3 일부와 GSH1 종결코돈 이후 500bp를 포함하는 PCR 산물을 획득한 후 각 PCR 산물을 동일한 몰비율로 S. cerevisiae CEN.PK2-1D 및 S. cerevisiae CC02-2544에 형질전환 하였다. PCR은 95°C에서 열변성 과정 5분, 53°C에서 결합과정 1분, 72°C에서 중합과정 1kb당 1분의 조건으로 수행하였으며 효모의 형질전환은 Geitz의 논문(Nucleic Acid Research, 20(6), 1425)을 변형한 리튬아세테이트 방법(Lithium acetate method)을 사용하였다. 구체적으로 O.D. 0.7 ~ 1.2 사이의 효모 세포를 리튬아세테이트/TE buffer로 2회 세척한 후, 상기 PCR산물들과 single stranded DNA (Sigma D-7656)을 함께 섞어 리튬아세테이트 ate/TE/40 % PEG buffer에서 30분간 30°C, 42°C에서 15분간 정치배양한 후 cell들을 우라실(Uracil)이 포함되지 않은 SC (2% glucose) agar plate에서 콜로니가 보일 때까지 배양하여 GSH1 G653M 변이 암호화 서열과 KlURA3 유전자가 도입된 균주를 획득하였다. 이후 KlURA3를 제거하기 위하여 각 균주를 2ml의 YPD에 overnight 배양 후 1/100 희석하여 0.1%의 5-FOA가 포함된 SC (2% glucose) agar plate에 도말하여 우라실(Uracil)마커가 제거된 S. cerevisiae CEN.PK2-1D GSH1 G653M 변이 균주 및 S. cerevisiae CC02-2544 GSH1 G653M 변이 균주를 제작하였다. 메티오닌 외에 다른 종류의 아미노산으로 치환된 GSH1 변이 단백질을 발현할 수 있는 균주도 서열번호 5 및 서열번호 6의 프라이머 서열상의 653번 메티오닌 코딩 서열을 다른 아미노산을 코딩하는 서열로 치환한 프라이머쌍을 이용한 점을 제외하고는 동일한 방식으로 제작하였다.Finally, in order to prepare a DNA fragment to be introduced into yeast, using the pWAL100-GSH1 (G653M) vector prepared above as a template and using the primers of SEQ ID NO: 4 and SEQ ID NO: 10 to include a methionine mutation coding sequence and a part of KlURA3 After obtaining a PCR product containing a part of KlURA3 and 500 bp after the GSH1 stop codon using the primers of SEQ ID NO: 11 and SEQ ID NO: 9 using the pWBR100-GSH1 vector as a template, each PCR product was obtained S. cerevisiae CEN.PK2-1D and S. cerevisiae CC02-2544 were transformed at the same molar ratio. PCR was performed under the conditions of 5 minutes for heat denaturation at 95°C, 1 minute for binding at 53°C, and 1 minute for polymerization at 72°C for 1 minute per kb. (6), 1425) modified lithium acetate method (Lithium acetate method) was used. Specifically, O.D. After washing yeast cells between 0.7 and 1.2 twice with lithium acetate/TE buffer, the PCR products and single stranded DNA (Sigma D-7656) were mixed together in lithium acetate ate/TE/40% PEG buffer for 30 minutes. After stationary incubation at 30°C and 42°C for 15 minutes, the cells were cultured on an SC (2% glucose) agar plate without Uracil until colonies were seen, and the GSH1 G653M mutation coding sequence and KlURA3 gene were introduced. strains were obtained. After that, in order to remove KlURA3, each strain was cultured in 2 ml of YPD overnight, diluted 1/100, spread on an SC (2% glucose) agar plate containing 0.1% 5-FOA, and the Uracil marker was removed. cerevisiae CEN.PK2-1D GSH1 G653M mutant strain and S. cerevisiae CC02-2544 GSH1 G653M mutant strain were prepared. A strain capable of expressing the GSH1 mutant protein substituted with an amino acid other than methionine also uses a primer pair in which the methionine coding sequence #653 on the primer sequences of SEQ ID NOs: 5 and 6 is substituted with a sequence encoding another amino acid. It was manufactured in the same way except for
프라이머primer 5' -> 3' 서열5' -> 3' sequence
F_BamHI_GSH1
(서열번호 4)F_BamHI_GSH1
(SEQ ID NO: 4) 		GGTAGGATCCATGGGACTCTTAGCTTTGGGCACGGTAGGATCCATGGGACTCTTAGCTTTTGGGCAC
R_GSH1_G653M
(서열번호 5)R_GSH1_G653M
(SEQ ID NO: 5) 		GTCAATTCCATTTTTGAATCGTCCAGTCAATTC CAT TTTTGAATCGTCCA
F_GSH1_G653M
(서열번호 6)F_GSH1_G653M
(SEQ ID NO: 6) 		ATTCAAAAATGGAATTGACATCCTTATTCAAAA ATG GAATTGACATCCTT
R_XhoI_GSH1
(서열번호 7)R_XhoI_GSH1
(SEQ ID NO: 7) 		ATGACTCGAGTTAACATTTGCTTTCTATTGAAGGCATGACTCGAGTTAACATTTGCTTTCTATTGAAGGC
F_SpeI_GSH1_DW
(서열번호 8)F_SpeI_GSH1_DW
(SEQ ID NO: 8) 		TAGAACTAGTACTCCTTTTATTTCGGTTGTGAATAGAACTAGTACTCCTTTTATTTCGGTTGTGAA
R_NcoI_GSH1_DW
(서열번호 9)R_NcoI_GSH1_DW
(SEQ ID NO: 9) 		GCTGCCATGGGAATAGTGTGAACCGATAACTGTGTGCTGCCATGGGAATAGTGTGAACCGATAACTGTGT
R_AL killer
(서열번호 10)R_AL killer
(SEQ ID NO: 10) 		GAGCAATGAACCCAATAACGAAATCTTGAGCAATGAACCCAATAACGAAATCTT
F_BR killer
(서열번호 11)F_BR killer
(SEQ ID NO: 11) 		CTTGACGTTCGTTCGACTGATGAGCTTGACGTTCGTTCGACTGATGAG
상기에서 제작된 각 균주를 26시간 배양하여 생산한 글루타치온 (GSH) 농도 및 함량을 측정한 결과를 표 2 및 표 3에 표시하였다.CC02-2544 균주에 GSH1 G653M 변이를 추가 도입(서열번호 13)한 결과, GSH 농도가 471.5 mg/L에서 548.5 mg/L로 77 mg/L 증가하였고, CEN.PK-1D 균주에 GSH1 G653M 변이(서열번호 3)를 도입한 결과 GSH 농도가 42 mg/L에서 100 mg/L로 58 mg/L 증가하였다.The results of measuring the glutathione (GSH) concentration and content produced by culturing each strain prepared above for 26 hours are shown in Tables 2 and 3. Additional introduction of GSH1 G653M mutation into the CC02-2544 strain (SEQ ID NO: 13) As a result, the GSH concentration increased by 77 mg/L from 471.5 mg/L to 548.5 mg/L, and as a result of introducing the GSH1 G653M mutation (SEQ ID NO: 3) into the CEN.PK-1D strain, the GSH concentration increased from 42 mg/L to 100 mg/L increased by 58 mg/L.
실시예 3-1: CC02-2544 균주에 GSH1 G653 변이 도입Example 3-1: GSH1 G653 mutation introduced into the CC02-2544 strain
S. cerevisiae CC02-2544S. cerevisiae CC02-2544
　 GSH 농도GSH concentration
(mg/L)(mg/L) 		GSH 함량GSH content
(%)(%) 		control 比control
GSH농도GSH concentration
(fold)(fold) 		control 比control
GSH함량GSH content
(fold)(fold)
WT (야생형)WT (wild type) 471.5471.5 3.23.2 1.001.00 1.001.00
GSH1 G653MGSH1 G653M 548.5548.5 3.83.8 1.161.16 1.191.19
GSH1 G653NGSH1 G653N 507.0507.0 3.33.3 1.081.08 1.031.03
GSH1 G653CGSH1 G653C 440.5440.5 3.23.2 0.930.93 1.001.00
GSH1 G653AGSH1 G653A 454.6454.6 3.23.2 0.960.96 1.001.00
GSH1 G653LGSH1 G653L 471.5471.5 3.23.2 1.001.00 1.001.00
GSH1 G653TGSH1 G653T 445.5445.5 3.03.0 0.940.94 0.940.94
GSH1 G653HGSH1 G653H 443.7443.7 3.03.0 0.940.94 0.930.93
GSH1 G653PGSH1 G653P 391.3391.3 2.92.9 0.830.83 0.900.90
GSH1 G653VGSH1 G653V 346.5346.5 2.42.4 0.730.73 0.750.75
실시예 3-2: CEN.PK-1D 균주에 GSH1 G653 변이 도입Example 3-2: GSH1 G653 mutation introduced into CEN.PK-1D strain
S. cerevisiae CEN.PK2-1DS. cerevisiae CEN.PK2-1D
　 GSH 농도GSH concentration
(mg/L)(mg/L) 		GSH 함량GSH content
(%)(%) 		control 比control
GSH농도GSH concentration
(fold)(fold) 		control 比control
GSHGSH
(fold)(fold)
WT (야생형)WT (wild type) 42.042.0 0.40.4 1.001.00 1.001.00
GSH1 G653MGSH1 G653M 100.0100.0 0.90.9 2.382.38 2.372.37
GSH1 G653LGSH1 G653L 53.053.0 0.50.5 1.261.26 1.271.27
GSH1 G653AGSH1 G653A 40.040.0 0.40.4 0.950.95 1.021.02
GSH1 G653CGSH1 G653C 42.042.0 0.40.4 1.001.00 1.021.02
GSH1 G653NGSH1 G653N 45.045.0 0.40.4 1.071.07 1.021.02
GSH1 G653HGSH1 G653H 39.039.0 0.40.4 0.950.95 0.960.96
GSH1 G653TGSH1 G653T 37.037.0 0.40.4 0.890.89 0.940.94
GSH1 G653VGSH1 G653V 36.036.0 0.30.3 0.860.86 0.870.87
GSH1 G653PGSH1 G653P 18.018.0 0.20.2 0.430.43 0.510.51
이를 통해 GSH1 단백질의 653번 글리신을 메티오닌으로 치환한 GSH1 변이체가 글루타치온 생산능을 크게 증가시키는 것임을 알 수 있다.Through this, it can be seen that the GSH1 mutant in which glycine 653 of the GSH1 protein is substituted with methionine greatly increases the glutathione production capacity.
이를 통해 본 출원에서 개발한 신규한 GSH1 변이체가 글루타치온 생산량 증가를 나타내는 것임을 확인할 수 있다. 또한, 본 출원의 GSH1 변이체를 포함하여 글루타치온을 고생산하는 효모, 이의 건조물, 추출물, 배양물, 파쇄물 및 생산된 글루타치온은, 항산화 효과, 해독 효과, 면역력 증강 효과를 가지는 것인바, 화장품용 조성물, 식품용 조성물, 사료용 조성물, 의약품 조성물 및 이의 제조에도 유용하게 사용될 수 있다.Through this, it can be confirmed that the novel GSH1 variant developed in the present application indicates an increase in glutathione production. In addition, the yeast that produces high glutathione, including the GSH1 mutant of the present application, its dried product, extract, culture, lysate, and produced glutathione have antioxidant effects, detoxification effects, and immunity enhancing effects, cosmetic compositions, It can be usefully used in a composition for food, a composition for feed, a pharmaceutical composition, and the preparation thereof.
참고예: GSH1 단백질의 C86 잔기 치환 실험Reference Example: C86 residue substitution experiment of GSH1 protein
GSH1 단백질의 86번째 시스테인 아미노산이 다른 아미노산으로 치환된 변이 단백질을 발현하도록 사카로마이세스 세레비지애(S. cerevisiae) CEN.PK2-1D 및 사카로마이세스 세레비지애(S. cerevisiae) CJ-5 균주의 변이 균주를 제작하여 글루타치온 생산량 증가 여부를 확인하고자 하였다. S. cerevisiae CEN.PK2-1D and S. cerevisiae CJ- It was attempted to confirm whether glutathione production increased by producing mutant strains of 5 strains.
사카로마이세스 세레비지애의 GSH1 단백질의 86번 시스테인을 아르기닌으로 치환한 균주를 제작하기 위하여 Lee TH, et al.(J. Microbiol. Biotechnol. (2006), 16(6), 979-982) 논문에 개시된 내용을 참고로 하여 pWAL100 및 pWBR100 플라스미드를 이용하였다. 구체적으로 CJ-5 균주의 genomic DNA를 주형(template)으로 하여 다음과 같이 PCR을 수행하였다. 서열번호 4와 서열번호 14의 프라이머를 이용한 PCR을 수행하여 N-terminal BamHI flanking 서열, GSH1 ORF의 개시코돈 및 C86R 변이 암호화 서열을 포함하는 GSH1 N-terminal일부 서열을 확보하고, 서열번호 15와 서열번호 7의 프라이머를 이용하여 C-terminal XhoI flanking 서열, GSH1 ORF 종결코돈 및 C86R 변이 암호화 서열을 포함하는 GSH1 C-terminal 일부 서열을 확보하였다. 이후 이 두 서열을 주형(template)으로 하여 서열번호 4와 서열번호 7를 이용하여 overlap PCR을 수행한 결과 86번째 시스테인이 아르기닌으로 치환된 GSH1 변이 단백질 암호화 서열, N-terminal BamHI, C-terminal XhoI 제한효소 서열을 포함하는 GSH1 ORF 절편을 확보하였다. 상기 ORF 절편은 BamHI 및 XhoI처리 후 동일한 효소로 처리한 pWAL100 벡터에 클로닝하여 pWAL100-GSH1(C86R)벡터를 제조하였다.In order to prepare a strain in which cysteine 86 of Saccharomyces cerevisiae GSH1 protein was substituted with arginine, Lee TH, et al. (J. Microbiol. Biotechnol. (2006), 16(6), 979-982) The pWAL100 and pWBR100 plasmids were used with reference to the contents disclosed in the paper. Specifically, PCR was performed as follows using the genomic DNA of the CJ-5 strain as a template. PCR was performed using the primers of SEQ ID NO: 4 and SEQ ID NO: 14 to secure a partial sequence of GSH1 N-terminal including the N-terminal BamHI flanking sequence, the initiation codon of the GSH1 ORF and the C86R variant coding sequence, and SEQ ID NO: 15 and the sequence A partial sequence of GSH1 C-terminal including the C-terminal XhoI flanking sequence, the GSH1 ORF stop codon and the C86R mutation coding sequence was obtained using the primer of No. 7. After performing overlap PCR using SEQ ID NO: 4 and SEQ ID NO: 7 using these two sequences as a template, the GSH1 mutant protein coding sequence in which the 86th cysteine was substituted with arginine, N-terminal BamHI, C-terminal XhoI A GSH1 ORF fragment containing a restriction enzyme sequence was obtained. The ORF fragment was cloned into the pWAL100 vector treated with the same enzyme after treatment with BamHI and XhoI to prepare a pWAL100-GSH1(C86R) vector.
또한, CJ-5 균주의 genomic DNA를 template으로 하여 서열번호 8과 서열번호 9의 프라이머를 이용한 PCR을 수행하여 N-terminal SpeI, C-terminal NcoI 제한효소 서열을 포함하는 GSH1 ORF 종결코돈 이후 500bp를 확보하고 SpeI, NcoI 제한효소를 처리하였다. 이후 동일한 제한효소를 처리한 pWBR100에 클로닝하여 pWBR100-GSH1벡터를 제조하였다.In addition, by performing PCR using the primers of SEQ ID NO: 8 and SEQ ID NO: 9 using the genomic DNA of the CJ-5 strain as a template, 500 bp after the GSH1 ORF stop codon including the N-terminal SpeI and C-terminal NcoI restriction enzyme sequences It was secured and treated with SpeI and NcoI restriction enzymes. After cloning into pWBR100 treated with the same restriction enzyme, pWBR100-GSH1 vector was prepared.
최종적으로 효모에 도입할 DNA 절편을 제작하기 위해 앞서 제작한 pWAL100-GSH1(C86R) 벡터를 주형(template)으로 하여 서열번호 4와 서열번호 10의 프라이머를 이용하여 아르기닌 변이 암호화서열과 KlURA3 일부를 포함하는 PCR 산물을 획득하고 pWBR100-GSH1벡터를 주형(template)으로 하여 서열번호 11과 서열번호 9의 프라이머를 이용하여 KlURA3 일부와 GSH1 종결코돈 이후 500bp를 포함하는 PCR 산물을 획득한 후 각 PCR 산물을 동일한 몰비율로 S. cerevisiae CEN.PK2-1D 및 S. cerevisiae CJ-5 에 형질전환 하였다. PCR은 95℃에서 열변성 과정 5분, 53℃에서 결합과정 1분, 72℃에서 중합과정 1kb당 1분의 조건으로 수행하였으며 효모의 형질전환은 Geitz의 논문(Nucleic Acid Research, 20(6), 1425)을 변형한 리튬아세테이트 방법(Lithium acetate method)을 사용하였다. 구체적으로 O.D. 0.7 ~ 1.2 사이의 효모 세포를 리튬아세테이트/TE buffer로 2회 세척 한 후, 상기 PCR산물들과 single stranded DNA (Sigma D-7656)을 함께 섞어 리튬아세테이트 ate/TE/40 % PEG buffer에서 30분간 30℃, 42℃에서 15분간 정치배양한 후 cell들을 우라실(Uracil)이 포함되지 않은 SC (2% glucose) agar plate에서 콜로니가 보일 때까지 배양하여 GSH1 C86R 변이 암호화 서열과 KlURA3 유전자가 도입된 균주를 획득하였다. 이후 KlURA3를 제거하기 위하여 각 균주를 2ml의 YPD에 overnight 배양 후 1/100 희석하여 0.1%의 5-FOA가 포함된 SC (2% glucose) agar plate에 도말하여 우라실(Uracil)마커가 제거된 S. cerevisiae CEN.PK2-1D GSH1 C86R 변이 균주 및 S. cerevisiae CJ-5 GSH1 C86R 변이 균주를 제작하였다. 아르기닌 외에 다른 아미노산으로 치환된 GSH1 변이 단백질을 발현할 수 있는 균주도 서열번호 14 및 서열번호 15의 프라이머 서열상의 86번 아르기닌 코딩 서열을 다른 아미노산을 코딩하는 서열로 치환한 프라이머쌍을 이용한 점을 제외하고는 동일한 방식으로 제작하였다.Finally, in order to prepare a DNA fragment to be introduced into yeast, using the pWAL100-GSH1 (C86R) vector prepared above as a template, the primers of SEQ ID NO: 4 and SEQ ID NO: 10 were used to include an arginine mutation coding sequence and a part of KlURA3 After obtaining a PCR product containing a part of KlURA3 and 500 bp after the GSH1 stop codon using the primers of SEQ ID NO: 11 and SEQ ID NO: 9 using the pWBR100-GSH1 vector as a template, each PCR product was obtained S. cerevisiae CEN.PK2-1D and S. cerevisiae CJ-5 were transformed at the same molar ratio. PCR was carried out under the conditions of 5 minutes for heat denaturation at 95°C, 1 minute for binding at 53°C, and 1 minute for polymerization at 72°C for 1 minute per kb. , 1425), a modified lithium acetate method was used. Specifically, O.D. After washing yeast cells between 0.7 and 1.2 twice with lithium acetate/TE buffer, the PCR products and single stranded DNA (Sigma D-7656) are mixed together in lithium acetate ate/TE/40% PEG buffer for 30 minutes After stationary culture at 30°C and 42°C for 15 minutes, the cells were cultured on an SC (2% glucose) agar plate without Uracil until colonies were seen, and the GSH1 C86R mutation coding sequence and KlURA3 gene were introduced has been obtained. After that, in order to remove KlURA3, each strain was cultured in 2 ml of YPD overnight, diluted 1/100, spread on an SC (2% glucose) agar plate containing 0.1% 5-FOA, and the Uracil marker was removed. cerevisiae CEN.PK2-1D GSH1 C86R mutant strain and S. cerevisiae CJ-5 GSH1 C86R mutant strain were prepared. A strain capable of expressing the GSH1 mutant protein substituted with other amino acids other than arginine also uses a primer pair in which the arginine coding sequence 86 on the primer sequences of SEQ ID NOs: 14 and 15 is substituted with a sequence encoding another amino acid. and produced in the same way.
프라이머primer 5'→3' 서열5'→3' sequence
F_BamHI_GSH1
(서열번호 4)F_BamHI_GSH1
(SEQ ID NO: 4) 		GGTAGGATCCATGGGACTCTTAGCTTTGGGCACGGTAGGATCCATGGGACTCTTAGCTTTTGGGCAC
R_GSH1_C86R
(서열번호 14)R_GSH1_C86R
(SEQ ID NO: 14) 		TTAGCCTCCCTAAGGGACGAATCCTTTAGCCTC CCT AAGGGACGAATCCT
F_GSH1_C86R
(서열번호 15)F_GSH1_C86R
(SEQ ID NO: 15) 		CGTCCCTTAGGGAGGCTAACGATGTCGTCCCTT AGG GAGGCTAACGATGT
R_XhoI_GSH1
(서열번호 7)R_XhoI_GSH1
(SEQ ID NO: 7) 		ATGACTCGAGTTAACATTTGCTTTCTATTGAAGGCATGACTCGAGTTAACATTTGCTTTCTATTGAAGGC
F_SpeI_GSH1_DW
(서열번호 8)F_SpeI_GSH1_DW
(SEQ ID NO: 8) 		TAGAACTAGTACTCCTTTTATTTCGGTTGTGAATAGAACTAGTACTCCTTTTATTTCGGTTGTGAA
R_NcoI_GSH1_DW
(서열번호 9)R_NcoI_GSH1_DW
(SEQ ID NO: 9) 		GCTGCCATGGGAATAGTGTGAACCGATAACTGTGTGCTGCCATGGGAATAGTGTGAACCGATAACTGTGT
R_AL killer
(서열번호 10)R_AL killer
(SEQ ID NO: 10) 		GAGCAATGAACCCAATAACGAAATCTTGAGCAATGAACCCAATAACGAAATCTT
F_BR killer
(서열번호 11)F_BR killer
(SEQ ID NO: 11) 		CTTGACGTTCGTTCGACTGATGAGCTTGACGTTCGTTCGACTGATGAG
상기에서 제작된 각 균주를 26시간 배양하여 생산한 글루타치온(GSH)농도를 측정한 결과를 표 5 및 표 6에 표시하였다. The results of measuring the glutathione (GSH) concentration produced by culturing each strain prepared above for 26 hours are shown in Tables 5 and 6.
Figure PCTKR2021012176-appb-img-000001
Figure PCTKR2021012176-appb-img-000001
Figure PCTKR2021012176-appb-img-000002
Figure PCTKR2021012176-appb-img-000002
실험 결과 GSH1 단백질의 86번 시스테인을 다른 아미노산으로 치환하는 경우 야생형 GSH1 단백질을 포함하는 경우보다 글루타치온 생산능이 증가하는 것을 확인할 수 있었다.As a result of the experiment, it was confirmed that when cysteine 86 of the GSH1 protein was substituted with another amino acid, the glutathione-producing ability increased compared to the case where the wild-type GSH1 protein was included.
이를 통해 GSH1 단백질의 86번 시스테인을 다른 아미노산으로 치환한 GSH1 변이체가 글루타치온 생산능을 크게 증가시키는 것임을 알 수 있다.From this, it can be seen that the GSH1 mutant in which the 86th cysteine of the GSH1 protein is substituted with another amino acid greatly increases the glutathione production capacity.
이상의 설명으로부터, 본 출원이 속하는 기술분야의 당업자는 본 출원이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로 이해해야만 한다. 본 출원의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 출원의 범위에 포함되는 것으로 해석되어야 한다.From the above description, those skilled in the art to which the present application pertains will be able to understand that the present application may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present application should be construed as including all changes or modifications derived from the meaning and scope of the claims to be described later rather than the above detailed description and their equivalent concepts to be included in the scope of the present application.
Figure PCTKR2021012176-appb-img-000003
Figure PCTKR2021012176-appb-img-000003
Figure PCTKR2021012176-appb-img-000004
Figure PCTKR2021012176-appb-img-000004
Figure PCTKR2021012176-appb-img-000005
Figure PCTKR2021012176-appb-img-000005
Figure PCTKR2021012176-appb-img-000006
Figure PCTKR2021012176-appb-img-000006

Claims (13)

  1. 서열번호 1의 아미노산 서열의 N-말단으로부터 653번째 위치에 상응하는 아미노산이 메티오닌으로 치환된, 글루타메이트-시스테인 리가아제(glutamate-cysteine ligase) 변이체.The amino acid corresponding to the 653th position from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is substituted with methionine, glutamate-cysteine ligase (glutamate-cysteine ligase) variant.
  2. 제1항에 있어서, 상기 653번째 위치에 상응하는 아미노산은 글리신인, 글루타메이트-시스테인 리가아제 변이체.The glutamate-cysteine ligase variant according to claim 1, wherein the amino acid corresponding to position 653 is glycine.
  3. 제1항에 있어서, 상기 변이체는 서열번호 1의 아미노산 서열과 80% 이상 및 100% 미만의 서열 상동성을 가지는 것인, 글루타메이트-시스테인 리가아제 변이체.The glutamate-cysteine ligase variant according to claim 1, wherein the variant has at least 80% and less than 100% sequence homology to the amino acid sequence of SEQ ID NO: 1.
  4. 제1항에 있어서, 상기 변이체는 서열번호 3의 아미노산 서열로 구성되는 것인, 글루타메이트-시스테인 리가아제 변이체.The glutamate-cysteine ligase variant according to claim 1, wherein the variant consists of the amino acid sequence of SEQ ID NO: 3.
  5. 제1항에 있어서, 상기 변이체는 추가로 86번째 위치에 상응하는 아미노산이 다른 아미노산으로 치환된, 글루타메이트-시스테인 리가아제(glutamate-cysteine ligase) 변이체.The glutamate-cysteine ligase variant according to claim 1, wherein the mutant is further substituted with another amino acid in the amino acid corresponding to the 86th position.
  6. 제5항에 있어서, 상기 변이체는 서열번호 13의 아미노산 서열로 구성되는 것인, 글루타메이트-시스테인 리가아제 변이체The glutamate-cysteine ligase variant according to claim 5, wherein the variant consists of the amino acid sequence of SEQ ID NO: 13.
  7. 제1항 내지 제6항 중 어느 한 항의 글루타메이트-시스테인 리가아제 변이체를 코딩하는 폴리뉴클레오티드.A polynucleotide encoding the glutamate-cysteine ligase variant of any one of claims 1-6.
  8. 제7항의 폴리뉴클레오티드를 포함하는 벡터.A vector comprising the polynucleotide of claim 7.
  9. 제1항 내지 제6항 중 어느 한 항의 글루타메이트-시스테인 리가아제 변이체; 상기 변이체를 코딩하는 폴리뉴클레오티드; 및 상기 폴리뉴클레오티드를 포함하는 벡터 중 어느 하나 이상을 포함하는, 글루타치온을 생산하는 미생물.The glutamate-cysteine ligase variant of any one of claims 1 to 6; a polynucleotide encoding the variant; And a microorganism producing glutathione, comprising any one or more of the vector containing the polynucleotide.
  10. 제9항에 있어서, 상기 미생물은 사카로마이세스 속 미생물인, 글루타치온을 생산하는 미생물.The microorganism according to claim 9, wherein the microorganism is a microorganism of the genus Saccharomyces.
  11. 제9항에 있어서, 상기 미생물은 사카로마이세스 세레비지애(Saccharomyces cerevisiae)인, 글루타치온을 생산하는 미생물.The microorganism according to claim 9, wherein the microorganism is Saccharomyces cerevisiae.
  12. 제1항 내지 제6항 중 어느 한 항의 글루타메이트-시스테인 리가아제 변이체; 상기 변이체를 코딩하는 폴리뉴클레오티드; 및 상기 폴리뉴클레오티드를 포함하는 벡터 중 어느 하나 이상을 포함하는 미생물을 배지에서 배양하는 단계를 포함하는, 글루타치온 생산 방법.The glutamate-cysteine ligase variant of any one of claims 1 to 6; a polynucleotide encoding the variant; And, glutathione production method comprising the step of culturing a microorganism comprising any one or more of the vector containing the polynucleotide in a medium.
  13. 제12항에 있어서, 상기 방법은 상기 배양된 미생물, 상기 미생물의 건조물, 상기 미생물의 추출물, 상기 미생물의 배양물, 및 상기 미생물의 파쇄물 중에서 선택된 하나 이상의 물질로부터 글루타치온을 회수하는 단계를 추가로 포함하는, 글루타치온 생산 방법.13. The method of claim 12, wherein the method further comprises recovering glutathione from at least one material selected from the cultured microorganism, the dried product of the microorganism, the extract of the microorganism, the culture of the microorganism, and the lysate of the microorganism. A method for producing glutathione.
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